Cardiovascular related uses of il-1beta antibodies and binding fragments thereof

ABSTRACT

Disclosed are methods for the reduction, prevention or treatment of cardiovascular events and/or cardiovascular diseases, including acute cardiovascular disease or chronic cardiovascular disease using anti-IL-1β binding molecules (e.g., IL-1β binding antibodies and fragments thereof). The present disclosure also relates to methods for prevention or treatment of cardiovascular events and/or cardiovascular diseases, including by reducing a cardiovascular event or disease.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/313,001, filed Mar. 11, 2010, U.S. Provisional Application No.61/252,571 filed Oct. 16, 2009, and U.S. Provisional Application No.61/182,679 filed May 29, 2009, the disclosures of which are incorporatedby reference herein in their entirety.

FIELD OF INVENTION

The present disclosure relates generally to IL-1β binding molecules(e.g., IL-1β binding antibodies and fragments thereof) for thereduction, prevention or treatment of cardiovascular events and/orcardiovascular diseases (e.g., acute cardiovascular disease or chroniccardiovascular disease).

BACKGROUND OF THE INVENTION

Inflammation has become a central theme in the pathogenesis ofcardiovascular disease over the past decade, and a wide range of cardiacdiseases has been associated with inflammation and cytokine modulation(Mehra et al., 2005, J. Leukocyte Biol. 78:805-818). Proinflammatorycytokines may be secreted by every nucleated cell type in themyocardium, including the cardiac myocyte, in response to various formsof stress/injury. They are elevated in conditions as diverse asinflammatory myocarditis, allograft rejection, cardiac ischemic states,congestive heart failure (CHF), and reperfusion injury.

IL-1β is a pro-inflammatory cytokine secreted by a number of differentcell types including monocytes and macrophages. When released as part ofan inflammatory reaction, IL-1β produces a range of biological effects,mainly mediated through induction of other inflammatory mediators suchas corticotrophin, platelet factor-4, prostaglandin E2 (PGE2), IL-6, andIL-8. IL-1β induces both local and systemic inflammatory effects throughthe activation of the IL-1 receptor found on almost all cell types. Theinterleukin-1 (IL-1) family of cytokines has been implicated in a numberof disease states. IL-1 family members include IL-1α, IL-1β, and IL-1Ra.Although related by their ability to bind to IL-1 receptors (IL-1R1 andIL-1R2), each of these cytokines is different, being expressed by adifferent gene and having a different primary amino acid sequence.Furthermore, the physiological activities of these cytokines can bedistinguished from each other.

SUMMARY OF THE INVENTION

The present disclosure relates generally to IL-1β binding molecules(e.g., IL-1β binding antibodies and fragments thereof) for thereduction, prevention or treatment of cardiovascular events and/orcardiovascular diseases, including acute cardiovascular disease orchronic cardiovascular disease. The present disclosure also relates tomethods for prevention or treatment of cardiovascular events and/orcardiovascular diseases, including by reducing a cardiovascular event ordisease.

The present disclosure provides methods of reducing a cardiovascularevent in a subject, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof, wherein the subject is a subject with ahistory of a previous cardiovascular event or a history of at least onerisk factor for cardiovascular disease, and wherein the cardiovascularevent is myocardial infarction, stroke, cardiovascular death, congestiveheart failure, cardiac arrest, acute coronary syndrome, angina, or arevascularization procedure.

The present disclosure provides methods of reducing a cardiovascularevent (e.g., delaying time to event, reducing likelihood or risk ofevent, preventing an event, reducing severity of event, reducing time torecovery) in a subject with a history of at least one risk factor forcardiovascular disease, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof, and wherein the cardiovascular event ismyocardial infarction, stroke, cardiovascular death, congestive heartfailure, cardiac arrest, acute coronary syndrome, angina, or arevascularization procedure.

The present disclosure also provides methods of reducing acardiovascular event (e.g., delaying time to event, reducing likelihoodor risk of event, preventing an event, reducing severity of event,reducing time to recovery) in a subject with a history of a previouscardiovascular event, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof, and wherein said cardiovascular event ismyocardial infarction, stroke, cardiovascular death, congestive heartfailure, cardiac arrest, acute coronary syndrome, angina or arevascularization procedure. In some embodiments, the previouscardiovascular event is a first cardiovascular event. In someembodiments, the previous or first cardiovascular event is selected fromthe group consisting of myocardial infarction, stroke, congestive heartfailure, acute coronary syndrome, angina and a revascularizationprocedure. In some embodiments, the previous or first cardiovascularevent is myocardial infarction or acute coronary syndrome. In someembodiments, the myocardial infarction is myocardial infarction with STelevation (e.g., ST-segment elevation myocardial infarction, STEMI). Insome embodiments, the myocardial infarction is myocardial infarctionwithout ST elevation (e.g., non-ST-segment elevation myocardialinfarction, NSTEMI). In some embodiments the presence or absence of STelevation is determined by electrocardiogram (e.g., ECG, EKG). In someembodiments, the method of reducing a cardiovascular event is a methodof reducing a second or subsequent cardiovascular event. In someembodiments, the cardiovascular event (e.g., second or subsequentcardiovascular event) is selected from the group consisting ofmyocardial infarction, stroke, cardiovascular death, congestive heartfailure, cardiac arrest, acute coronary syndrome, angina and arevascularization procedure. In some embodiments, the firstcardiovascular event and second cardiovascular event are the same typesof cardiovascular events. In some embodiments, the first cardiovascularevent and second cardiovascular event are different types ofcardiovascular events.

In some embodiments, the revascularization procedure is a coronary,carotid or peripheral arterial revascularization procedure. In someembodiments, the coronary, carotid or peripheral arterialrevascularization procedure is a percutaneous coronary intervention(PCI), a stent implant, coronary artery bypass graft (CABG), carotidendarterectomy, peripheral vascular disease bypass surgery, orperipheral angioplasty surgery.

In some embodiments, said subject also has a history of at least onerisk factor for cardiovascular disease. In some embodiments, the riskfactor is manifest coronary heart disease, coronary artery disease,thrombosis, transient ischaemic attack, left ventricular hypertrophy,arteriosclerosis, restenosis, tobacco smoking or peripheral vasculardisease. In some embodiments, the risk factor is elevated triglycerides,systemic inflammation, high blood phosphorus levels, high parathyroidhormone levels, microalbuminuria, or high homocysteine levels. In someembodiments, the risk factor is obesity, hyperglycemia, chronic renalfailure, high blood glucose, chronic kidney disease, or metabolicsyndrome. In some embodiments, the risk factor is end stage renaldisease. In some embodiments, the risk factor is hypertension,dyslipidemia, hyperlipidemia, elevated total cholesterol, elevated LDLcholesterol, or low HDL cholesterol or atherosclerosis. In someembodiments, the hypertension is manifested as a blood pressure ofgreater than or equal to 180/110 mm Hg. In some other embodiments, thehypertension is mild-to-moderate, with systolic blood pressure (SBP) of140 to 180 mm Hg and/or diastolic blood pressure (DBP) of 90 to 110 mmHg.

In some embodiments, the subject has elevated levels of C-reactiveprotein (CRP).

In some embodiments, the subject is older than 55 years.

In some embodiments, the subject is older than 65 years.

In some embodiments, the subject is non-hypertensive.

In some embodiments, the subject has poorly controlled hypertension.

In some embodiments, the subject has an arrhythmia.

In some embodiments, the subject has a “Type A” personality.

In some embodiments, the subject has a sedentary lifestyle.

In some embodiments, the subject has diabetes mellitus. In someembodiments, said diabetes mellitus is Type 2 diabetes.

In some embodiments, the subject has a history of two or more said riskfactors.

In some embodiments, the subject has a history of three or more saidrisk factors.

In some embodiments, administering said therapeutically effective amountof an anti-IL-1β binding antibody or binding fragment thereof issufficient to achieve a decrease in CRP levels.

The present disclosure also provides methods of reducing mortalityfollowing a cardiovascular event in a subject, comprising administeringto said subject a therapeutically effective amount of an anti-IL-1βbinding antibody or binding fragment thereof.

In some embodiments, the cardiovascular event is myocardial infarction,stroke, congestive heart failure, acute coronary syndrome, angina or arevascularization procedure. In some embodiments, the cardiovascularevent is myocardial infarction or acute coronary syndrome. In someembodiments, the myocardial infarction is myocardial infarction with STelevation (e.g., ST-segment elevation myocardial infarction, STEMI). Insome embodiments, the myocardial infarction is myocardial infarctionwithout ST elevation (e.g., non-ST-segment elevation myocardialinfarction, NSTEMI). In some embodiments the presence or absence of STelevation is determined by electrocardiogram (e.g., ECG, EKG). In someembodiments, the revascularization procedure is a coronary, carotid orperipheral arterial revascularization procedure. In some embodiments,the coronary, carotid or peripheral arterial revascularization procedureis a percutaneous coronary intervention (PCI), a stent implant, coronaryartery bypass graft (CABG), carotid endarterectomy, peripheral vasculardisease bypass surgery, or peripheral angioplasty surgery.

In some embodiments, the subject does not have Type 2 diabetes.

In some embodiments, the subject has survived a previous cardiovascularevent of myocardial infarction or stroke.

In some embodiments, the occurrence of said cardiovascular event is areoccurrence of a cardiovascular event of myocardial infarction orstroke.

In some embodiments, the subject has a history of one or more riskfactors for cardiovascular disease. In some embodiments, the risk factoris manifest coronary heart disease, coronary artery disease, thrombosis,transient ischaemic attack, left ventricular hypertrophy,arteriosclerosis, restenosis, tobacco smoking or peripheral vasculardisease. In some embodiments, the risk factor is elevated triglycerides,systemic inflammation, high blood phosphorus levels, high parathyroidhormone levels, microalbuminuria, or high homocysteine levels. In someembodiments, the risk factor is obesity, hyperglycemia, chronic renalfailure, high blood glucose, chronic kidney disease, or metabolicsyndrome. In some embodiments, the risk factor is end stage renaldisease. In some embodiments, the risk factor is hypertension,dyslipidemia, hyperlipidemia, elevated total cholesterol, elevated LDLcholesterol, or low HDL cholesterol or atherosclerosis. In someembodiments, the hypertension is manifested as a blood pressure ofgreater than or equal to 180/110 mm Hg. In some other embodiments, thehypertension is mild-to-moderate, with systolic blood pressure (SBP) of140 to 180 mm Hg and/or diastolic blood pressure (DBP) of 90 to 110 mmHg.

In some embodiments, the subject is non-hypertensive.

In some embodiments, the subject has poorly controlled hypertension.

In some embodiments, the subject has an arrhythmia.

In some embodiments, the subject has a “Type A” personality.

In some embodiments, the subject has a sedentary lifestyle.

In some embodiments, the subject has a history of two or more said riskfactors.

In some embodiments, the subject has a history of three or more saidrisk factors.

In some embodiments, the subject is a patient with cardiovasculardisease, including acute cardiovascular disease (e.g., not associatedwith congestive heart failure) or chronic cardiovascular disease (e.g.,associated with multiple risk factors for atherosclerotic cardiovasculardisease).

In some embodiments, administering said therapeutically effective amountof an anti-IL-1β binding antibody or binding fragment thereof issufficient to achieve a decrease in CRP levels.

The present disclosure also provides methods of reducing acardiovascular event in a subject with a history of at least one riskfactor for cardiovascular disease, comprising administering to saidsubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof, and wherein said risk factor isnot Type 2 diabetes, obesity, hyperglycemia, dyslipidemia,hyperlipidemia, chronic renal failure, high blood glucose, chronickidney disease, hypertension, atherosclerosis or metabolic syndrome.

In some embodiments, the cardiovascular event is myocardial infarction,stroke, cardiac arrest, congestive heart failure, cardiovascular death,acute coronary syndrome (e.g., diagnosed), angina or a revascularizationprocedure. In some embodiments, the revascularization procedure is acoronary, carotid or peripheral arterial revascularization procedure. Insome embodiments, the coronary, carotid or peripheral arterialrevascularization procedure is a percutaneous coronary intervention(PCI), a stent implant, coronary artery bypass graft (CABG), carotidendarterectomy, peripheral vascular disease bypass surgery, orperipheral angioplasty surgery.

In some embodiments, the risk factor is manifest coronary heart disease,coronary artery disease, thrombosis, transient ischaemic attack, leftventricular hypertrophy, arteriosclerosis, restenosis, tobacco smokingor peripheral vascular disease. In some embodiments, the risk factor iselevated triglycerides, systemic inflammation, high blood phosphoruslevels, high parathyroid hormone levels, microalbuminuria, or highhomocysteine levels.

In some embodiments, the subject has elevated levels of C-reactiveprotein (CRP).

In some embodiments, the subject is older than 55 years.

In some embodiments, the subject is older than 65 years.

In some embodiments, the subject has a history of two or more said riskfactors.

In some embodiments, the subject has a history of three or more saidrisk factors.

In some embodiments, the subject is a patient with cardiovasculardisease, including acute cardiovascular disease (e.g., not associatedwith congestive heart failure) or chronic cardiovascular disease (e.g.,associated with multiple risk factors for atherosclerotic cardiovasculardisease).

In some embodiments, administering said therapeutically effective amountof an anti-IL-1β binding antibody or binding fragment thereof issufficient to achieve a decrease in CRP levels.

The present disclosure also provides methods of treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof and at least one other pharmaceuticalcomposition comprising an active agent other than an IL-1β antibody orfragment.

In some embodiments, the cardiovascular event is myocardial infarctionor acute coronary syndrome. In some embodiments, the myocardialinfarction is myocardial infarction with ST elevation (e.g., ST-segmentelevation myocardial infarction, STEMI). In some embodiments, themyocardial infarction is myocardial infarction without ST elevation(e.g., non-ST-segment elevation myocardial infarction, NSTEMI). In someembodiments the presence or absence of ST elevation is determined byelectrocardiogram (e.g., ECG, EKG).

In some embodiments, the active agent of said at least one otherpharmaceutical composition is a cholesterol lowering agent, a statin, anHMG-CoA reductase inhibitor, a calcium channel blocker, a beta blocker,an antihypertensive, a diuretic, aspirin, niacin, anangiotensin-converting enzyme (ACE) inhibitor, an angiotensin IIreceptor blocker, a vasodilator, an anticoagulant, a inhibitor ofplatelet aggregation, a thrombolytic or digitalis.

The present disclosure also provides methods of treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof and (e.g., in conjunction with) arevascularization procedure.

In some embodiments, the cardiovascular event is myocardial infarctionor acute coronary syndrome. In some embodiments, the myocardialinfarction is myocardial infarction with ST elevation (e.g., ST-segmentelevation myocardial infarction, STEMI). In some embodiments, themyocardial infarction is myocardial infarction without ST elevation(e.g., non-ST-segment elevation myocardial infarction, NSTEMI). In someembodiments the presence or absence of ST elevation is determined byelectrocardiogram (e.g., ECG, EKG).

In some embodiments, the revascularization procedure is a coronary,carotid or peripheral arterial revascularization procedure.

The present disclosure also provides methods of treating cardiovasculardisease, including, for example, acute cardiovascular disease or chroniccardiovascular disease, in a subject, comprising administering to saidsubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof and (e.g., in conjunction with) arevascularization procedure.

In some embodiments, administering said therapeutically effective amountof an anti-IL-1β binding antibody or binding fragment thereof issufficient to achieve a reduction in the relative risk (e.g., lowerrisk, frequency, incidence, severity) of MACE (major adverse cardiacevent, e.g., myocardial infarction, stroke, death, such as CV death,and/or composite thereof), including, for example, in patients withcardiovascular disease, such as acute cardiovascular disease or chroniccardiovascular disease, or in patients with multiple risk factors foratherosclerotic cardiovascular disease (e.g., age 55, age 65, plus oneor more of: CABG, NSTEMI, hypertension, elevated cholesterol or onstatins, elevated CRP, prior history of Myocardial infarction/stroke noless than 6 months, prior history of ACS or TIA, smoking, history ofPCI, type 2 diabetes).

In some embodiments, administering said therapeutically effective amountof an anti-IL-1β binding antibody or binding fragment thereof issufficient to achieve an in time to first MACE event, revascularizationprocedures (e.g., CABG), all cause mortality, peripheral vasculardisease, first documented angina endpoint, hospitalization forcongestive heart failure (CHF), decrease in number of hospital visits,duration of hospital stay, rehospitalization for ischemic events (e.g.,angina and/or CHF), infarct size, diastolic volume, ejection fraction oruse of diuretics.

In some embodiments, administering said therapeutically effective amountof an anti-IL-1β binding antibody or binding fragment thereof issufficient to achieve plaque regression, plaque stabilization and/orinhibition of plaque rupture.

In some embodiments, administering said therapeutically effective amountof an anti-IL-1β binding antibody or binding fragment thereof issufficient to achieve a decrease in CRP levels, BNP levels, troponinlevels, C-peptide levels, LDL levels, blood pressure or blood sugar(HbA1c).

In some embodiments, administering said therapeutically effective amountof an anti-IL-1β binding antibody or binding fragment thereof issufficient to achieve a decrease or no increase in SAE, malignancy,hypoglycemia, serious infection rate, infection rate, immunogenicity orheart failure.

The present disclosure also provides methods of reducing restenosis in asubject following a revascularization procedure, comprisingadministering to said subject a therapeutically effective amount of ananti-IL-1β binding antibody or binding fragment thereof.

In some embodiments, the revascularization procedure is a coronary,carotid or peripheral arterial revascularization procedure.

In some embodiments, administering said therapeutically effective amountof an anti-IL-1β binding antibody or binding fragment thereof issufficient to achieve a decrease in CRP levels.

The present disclosure also provides methods of treating acutehypertension in a subject comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof and one or more antihypertensive agents. Insome embodiments, the subject has a blood pressure of greater than orequal to 180/110 mm Hg. In some other embodiments, the subject hasmild-to-moderate hypertension, with systolic blood pressure (SBP) of 140to 180 mm Hg and/or diastolic blood pressure (DBP) of 90 to 110 mm Hg.In some embodiments, the antihypertensive agent is administeredintravenously. In some embodiments, the antihypertensive agent isselected from the group consisting of alpha/beta-adrenergic blockingagents, angiotensin-converting enzyme inhibitors, angiotensin IIreceptor antagonists, antiadrenergic agents, beta-adrenergic blockingagents, calcium-channel blocking agents, diuretics, and vasodilators. Insome embodiments, the antihypertensive agent is carvedilol, labetalol,benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril,perindopril, quinapril, ramipril, trandolapril, candesartan, eprosartan,irbesartan, losartan, telmisartan, valsartan, clonidine, doxazosin,guanabenz, guanadrel, guanethidine, guanfacine, mecamylamine,methyldopa, prazosin, reserpine, terazosin, acebutolol, atenolol,betaxolol, bisoprolol, carteolol, metoprolol, nadolol, penbutolol,pindolol, propranolol, timolol, amlodipine, diltiazem, felodipine,isradipine, nicardipine, nifedipine, nisoldipine, verapamil, amiloride,benzthiazide, chlorothiazide, chlorthalidone, furosemide,hydrochlorothiazide, indapamide, metolazone, polythiazide,spironolactone, torsemide, trichlormethiazide, hydralazine,nitroglycerin, sodium nitroprusside, clevidipine or minoxidil. In someembodiments, the antihypertensive agent is labetalol, metoprolol,hydralazine, nitroglycerin, nicardipine, sodium nitroprusside orclevidipine.

The present disclosure also provides methods of reducing, preventing ortreating a cardiovascular event or disease (e.g., acute cardiovasculardisease or chronic cardiovascular disease) in a subject comprisingadministering to the subject an anti-IL-1β binding antibody or bindingfragment thereof in combination with (e.g., in conjunction with) (e.g.,before, during or after) a medical or surgical intervention. Suchantibodies may be administered in therapeutically effective amounts.Such interventions may be therapeutically effective. In someembodiments, a medical intervention is an active agent, such as a drugor a biologic, including, for example, any one or more of the activeagents described herein. In some embodiments, a medical intervention isan out-patient medical treatment or procedure. In some embodiments, amedical intervention is an in-patient hospitalization. In someembodiments, a surgical intervention is a revascularization procedure,including, for example, any one or more of the revascularizationprocedures described herein. In some embodiments, a surgicalintervention involves a heart valve repair or replacement, coronarybypass surgery, heart transplant or heart pump. In some embodiments, asurgical intervention involves a biventricular cardiac pacemaker,internal cardiac defibrillator (ICD) or myectomy. In some embodiments, amedical intervention is smoking cessation medication or smokingcessation counseling.

The present disclosure also provides methods of reducing acardiovascular event in a subject with a history of at least one riskfactor for cardiovascular disease, comprising (a) identifying,diagnosing or selecting the subject with the history of at least onerisk factor for cardiovascular disease and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof, and wherein the cardiovascularevent is myocardial infarction, stroke, cardiovascular death, congestiveheart failure, cardiac arrest, acute coronary syndrome, angina, or arevascularization procedure.

The present disclosure also provides methods of reducing acardiovascular event in a subject with a history of a previouscardiovascular event, comprising (a) identifying, diagnosing orselecting the subject with the history of the previous cardiovascularevent and (b) administering to the subject a therapeutically effectiveamount of an anti-IL-1β binding antibody or binding fragment thereof,and wherein the cardiovascular event is myocardial infarction, stroke,acute coronary syndrome, angina or a revascularization procedure. Insome embodiments, the previous cardiovascular event is a firstcardiovascular event. In some embodiments, the previous or firstcardiovascular event is selected from the group consisting of myocardialinfarction, stroke, congestive heart failure, acute coronary syndrome,angina and a revascularization procedure. In some embodiments, theprevious or first cardiovascular event is myocardial infarction or acutecoronary syndrome. In some embodiments, the myocardial infarction ismyocardial infarction with ST elevation (e.g., ST-segment elevationmyocardial infarction, STEMI). In some embodiments, the myocardialinfarction is myocardial infarction without ST elevation (e.g.,non-ST-segment elevation myocardial infarction, NSTEMI). In someembodiments the presence or absence of ST elevation is determined byelectrocardiogram (e.g., ECG, EKG). In some embodiments, the method ofreducing a cardiovascular event is a method of reducing a second orsubsequent cardiovascular event. In some embodiments, the cardiovascularevent (e.g., second or subsequent cardiovascular event) is selected fromthe group consisting of myocardial infarction, stroke, cardiovasculardeath, congestive heart failure, cardiac arrest, acute coronarysyndrome, angina and a revascularization procedure. In some embodiments,the first cardiovascular event and second cardiovascular event are thesame types of cardiovascular events. In some embodiments, the firstcardiovascular event and second cardiovascular event are different typesof cardiovascular events.

The present disclosure also provides methods of reducing mortalityfollowing a cardiovascular event in a subject, comprising (a)identifying, diagnosing or selecting the subject having thecardiovascular event and (b) administering to the subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof. In some embodiments, the cardiovascular eventis selected from the group consisting of myocardial infarction, stroke,congestive heart failure, acute coronary syndrome, angina and arevascularization procedure. In some embodiments, the cardiovascularevent is myocardial infarction or acute coronary syndrome. In someembodiments, the myocardial infarction is myocardial infarction with STelevation (e.g., ST-segment elevation myocardial infarction, STEMI). Insome embodiments, the myocardial infarction is myocardial infarctionwithout ST elevation (e.g., non-ST-segment elevation myocardialinfarction, NSTEMI). In some embodiments the presence or absence of STelevation is determined by electrocardiogram (e.g., ECG, EKG).

The present disclosure also provides methods of reducing acardiovascular event in a subject with a history of at least one riskfactor for cardiovascular disease, comprising (a) identifying,diagnosing or selecting the subject with the history of at least onerisk factor for cardiovascular disease and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof, and wherein the risk factor is notType 2 diabetes, obesity, hyperglycemia, dyslipidemia, hyperlipidemia,chronic renal failure, high blood glucose, chronic kidney disease,hypertension, atherosclerosis or metabolic syndrome. In someembodiments, the cardiovascular event is selected from the groupconsisting of myocardial infarction, stroke, cardiovascular death,congestive heart failure, cardiac arrest, acute coronary syndrome,angina and a revascularization procedure.

The present disclosure also provides methods of treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising (a) identifying, diagnosing or selectingthe subject with the cardiovascular event and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof and at least one otherpharmaceutical composition comprising an active agent other than anIL-1β antibody or fragment. In some embodiments, the previous or firstcardiovascular event is myocardial infarction or acute coronarysyndrome. In some embodiments, the myocardial infarction is myocardialinfarction with ST elevation (e.g., ST-segment elevation myocardialinfarction, STEMI). In some embodiments, the myocardial infarction ismyocardial infarction without ST elevation (e.g., non-ST-segmentelevation myocardial infarction, NSTEMI). In some embodiments thepresence or absence of ST elevation is determined by electrocardiogram(e.g., ECG, EKG).

The present disclosure also provides methods for treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising (a) identifying, diagnosing or selectingthe subject with the cardiovascular event and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof and (e.g., in conjunction with) arevascularization procedure. In some embodiments, the previous or firstcardiovascular event is myocardial infarction or acute coronarysyndrome. In some embodiments, the myocardial infarction is myocardialinfarction with ST elevation (e.g., ST-segment elevation myocardialinfarction, STEMI). In some embodiments, the myocardial infarction ismyocardial infarction without ST elevation (e.g., non-ST-segmentelevation myocardial infarction, NSTEMI). In some embodiments thepresence or absence of ST elevation is determined by electrocardiogram(e.g., ECG, EKG).

The present disclosure also provides methods of reducing restenosis in asubject following a revascularization procedure, comprising (a)identifying, diagnosing or selecting the subject with therevascularization procedure and (b) administering to the subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof.

The present disclosure also provides methods of treating acutehypertension in a subject comprising (a) identifying, diagnosing orselecting the subject with acute hypertension and (b) administering tothe subject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof and one or more antihypertensiveagents. In some embodiments, the hypertension is manifested as a bloodpressure of greater than or equal to 180/110 mm Hg. In some otherembodiments, the hypertension is mild-to-moderate, with systolic bloodpressure (SBP) of 140 to 180 mm Hg and/or diastolic blood pressure (DBP)of 90 to 110 mm Hg.

In any and/or all of the aforementioned embodiments, administering saidtherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof may be sufficient to achieve a decrease in CRPlevels.

The present disclosure also provides pharmaceutical compositions for usein any and/or all of the aforementioned methods, including for example,for the reduction, prevention or treatment of cardiovascular eventsand/or cardiovascular diseases, including acute cardiovascular diseaseor chronic cardiovascular disease, by administering a therapeuticallyeffective amount of an anti-IL-1β binding antibody or binding fragmentthereof.

Various methods and pharmaceutical compositions are provided herein,including for example, those described above. The present disclosurefurther provides IL-1β binding antibodies and binding fragments thereof,as well as suitable dose amounts and dosing regimens that may be used inor with any and/or all of the aforementioned methods and pharmaceuticalcompositions.

In some embodiments of any and/or all of the methods and pharmaceuticalcompositions described above, the antibody or fragment binds to humanIL-1β with a dissociation constant of about 1 nM or less. In someembodiments, the antibody or fragment binds to human IL-1β with adissociation constant of about 500 pM or less. In some embodiments, theanti-IL-1β binding antibody or binding fragment thereof binds to humanIL-1β with a dissociation constant of about 250 pM or less. In someembodiments, the anti-IL-1β binding antibody or binding fragment thereofbinds to human IL-1β with a dissociation constant of about 100 pM orless. In some embodiments of any of the methods described above, theanti-IL-1β binding antibody or binding fragment thereof binds to humanIL-1β with a dissociation constant of about 50 pM or less. In someembodiments of any of the methods described above, the anti-IL-1βbinding antibody or binding fragment thereof binds to human IL-1β with adissociation constant of about 5 pM or less. In some embodiments, theanti-IL-1β binding antibody or binding fragment thereof binds to humanIL-1β with a dissociation constant of about 1 pM or less. In someembodiments, the anti-IL-1β binding antibody or binding fragment thereofbinds to human IL-1β with a dissociation constant of about 0.3 pM orless.

In some embodiments of any and/or all of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof is aneutralizing antibody.

In some embodiments of any and/or all of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof binds to anIL-1β epitope such that the bound antibody or fragment substantiallypermits the binding of IL-1β to IL-1 receptor I (IL-1R1).

In some embodiments of any and/or all of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof does notdetectably bind to IL-1α, IL-1R or IL-1Ra.

In some embodiments of any and/or all of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof competeswith the binding of an antibody having the light chain variable regionof SEQ ID NO:1 and the heavy chain variable region of SEQ ID NO:2. Insome embodiments of any and/or all of the methods described above, theanti-IL-1β binding antibody or binding fragment thereof binds to anepitope that is the same or substantially the same as an epitope that isbound by an antibody having the light chain variable region of SEQ IDNO:1 and the heavy chain variable region of SEQ ID NO:2. In someembodiments of any and/or all of the methods described above, theanti-IL-1β binding antibody or binding fragment thereof comprises alight chain variable region of SEQ ID NO:1 and a heavy chain variableregion of SEQ ID NO:2.

In some embodiments of any and/or all of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof binds to anepitope incorporating Glu64 of IL-1β.

In some embodiments of any and/or all of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof binds toamino acids 1-34 of the N terminus of IL-1β.

In some embodiments of any and/or all of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof is humanizedor human.

In some embodiments of any and/or all of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof isadministered in one or more doses of 3 mg/kg of antibody or fragment. Insome embodiments, the anti-IL-1β binding antibody or binding fragmentthereof is administered in one or more doses of 1 mg/kg or less ofantibody or fragment. In some embodiments, the anti-IL-1β bindingantibody or binding fragment thereof is administered in one or moredoses of 0.3 mg/kg or less of antibody or fragment. In some embodiments,the anti-IL-1β binding antibody or binding fragment thereof isadministered in one or more doses of 0.1 mg/kg or less of antibody orfragment. In some embodiments, the anti-IL-1β binding antibody orbinding fragment thereof is administered in one or more doses of 0.03mg/kg or less of antibody or fragment. In some embodiments, the one ormore doses are at least 0.01 mg/kg of antibody or fragment. In someembodiments of any of the methods described above, the anti-IL-1βbinding antibody or binding fragment thereof is administered in one ormore doses of 0.03 mg/kg to 1 mg/kg.

In some embodiments of any and/or all of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof isadministered as a fixed dose, independent of a dose per subject weightratio. In some embodiments, the anti-IL-1β binding antibody or bindingfragment thereof is administered in one or more doses of 100 mg or lessof antibody or fragment. In some embodiments, the anti-IL-1β bindingantibody or binding fragment thereof is administered in one or moredoses of 25 mg or less of antibody or fragment In some embodiments, theanti-IL-1β binding antibody or binding fragment thereof is administeredin one or more doses of 10 mg or less of antibody or fragment. In someembodiments, the anti-IL-1β binding antibody or binding fragment thereofis administered in one or more doses of at least 0.5 mg of antibody orfragment. In some embodiments, the anti-IL-1β binding antibody orbinding fragment thereof is administered in one or more doses of 1 mg to100 mg of antibody or fragment. In some embodiments, said fixed dose ofanti-IL-1β binding antibody or binding fragment thereof is administeredusing a pre-filled syringe or delivery device.

In some embodiments of any and/or all of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof isadministered by subcutaneous, intravenous or intramuscular injection.

In some embodiments of any and/or all of the methods described above,administration of an initial dose of anti-IL-1β binding antibody orbinding fragment thereof is followed by the administration of one ormore subsequent doses. In some embodiments, said initial dose and one ormore subsequent doses are administered at an interval of about onceevery week to about once every 12 months. In some embodiments, saidinitial dose and one or more subsequent doses are administered at aninterval of about once every two weeks to about once every 6 months. Insome embodiments, said initial dose and one or more subsequent doses areadministered at an interval of about once every month to about onceevery 6 months. In some embodiments, said initial dose and one or moresubsequent doses are administered at an interval of about once everymonth to about once every 3 months. In some embodiments, said initialdose and one or more subsequent doses are administered at an interval ofabout once every 3 months to about once every 6 months.

In some embodiments of any and/or all of the aforementioned methodsdosing regimens are provided, wherein the dosing regimen comprises morethan one dosing interval for administration of an IL-1β binding antibodyor binding fragment thereof. In some embodiments, the dosage regimencomprises at least two (e.g., two, three, four, five, six) differentdosing intervals for administration of the IL-1β antibody or fragmentthereof. In some embodiments, the dosage regimen comprises two differentdosing intervals for administration of the IL-1β antibody or fragmentthereof. In some embodiments, the dosing regimen comprises two differentdosing intervals for administration of the IL-1β binding antibody orbinding fragment thereof, wherein a first dosing interval comprisesadministration of one or more doses of the IL-1β antibody or fragmentthereof and a second dosing interval comprises administration of one ormore doses of the IL-1β antibody or fragment thereof, and wherein thefirst dosing interval is shorter in time than the second dosinginterval. For example, the first dosing interval may be days or weeks,and the second dosing interval may be months. In some embodiments, thefirst dosing interval is about 5 days to about 28 days, about 7 days toabout 21 days, about 12 days to about 16 days, or about 14 days. In someembodiments, the second dosing interval is about 1 month to about 3months, about 1 month to about 2 months, or about 1 month. In someembodiments, the first dosing interval is about 7 days and the seconddosing interval is about 1 month.

In some embodiments, administration of an initial dose of anti-IL-1βbinding antibody or binding fragment thereof is followed byadministration of one or more subsequent doses, and wherein the dosingintervals between administration of the initial dose and a second dose,and the second dose and a third dose are about 7 days to about 21 days,and wherein the dosing intervals between administration of subsequentdoses is about 1 month to about 3 months. In some embodiments, thedosing intervals between administration of the initial dose and a seconddose, and the second dose and a third dose are about 12 to 16 days, andthe dosing intervals between administration of subsequent doses is about1 month to about 2 months. In some embodiments, the dosing intervalsbetween administration of the initial dose and a second dose, and thesecond dose and a third dose are about 14 days, and the dosing intervalsbetween administration of subsequent doses is about 1 month.

In some preferred embodiments of any and/or all of the aforementionedmethods, dose amounts and/or dosing regimens, the IL-1β binding antibodyor binding fragment thereof (e.g., therapeutically effective amount ofan anti-IL-1β binding antibody or binding fragment thereof) is firstadministered within 1 week of the cardiovascular event, within 96 hoursof the cardiovascular event, within 72 hours of the cardiovascularevent, within 48 hours of the cardiovascular event, within 24 hours ofthe cardiovascular event, or within 12 hours of the cardiovascularevent.

In some embodiments of any and/or all of the methods described above,administration of an initial dose of the anti-IL-1β binding antibody orbinding fragment thereof is followed by the administration of one ormore subsequent doses, and wherein said one or more subsequent doses arein an amount that is approximately the same or less than the initialdose.

In some embodiments of any and/or all of the methods described above,administration of an initial dose of the anti-IL-1β binding antibody orbinding fragment thereof is followed by the administration of one ormore subsequent doses, and wherein at least one of the subsequent dosesis in an amount that is more than the initial dose.

In some embodiments of any and/or all of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof thereof hasa lower IC₅₀ than an IL-1β receptor antagonist in a human whole bloodIL-1β inhibition assay that measures IL-1β induced production of IL-8.In some embodiments, the IL-1β receptor antagonist is anakinra.

In some embodiments, any and/or all of the methods described above mayfurther comprise administering at least one other pharmaceuticalcomposition comprising an active agent other than an anti-IL-1β bindingantibody or binding fragment thereof. In some embodiments, the activeagent of said at least one other pharmaceutical composition is acholesterol lowering agent. In some embodiments, the active agent ofsaid at least one other pharmaceutical composition is a statin or anHMG-CoA reductase inhibitor (e.g., lovastatin, pravastatin, simvastatin,fluvastatin, atorvastatin, cerivastatin, mevastatin, pitavastatin,rosuvastatin or mixtures thereof or mixtures with Ezetimibe, niacin,Amlodipine Besylate). In some embodiments, the active agent of said atleast one other pharmaceutical composition is a calcium channel blocker(e.g., amlodipine, diltiazem, nifedipine, nicardipine, verapamil) or abeta blocker (e.g., esmolol, metoprolol, nadolol, penbutolol). In someembodiments, the active agent of said at least one other pharmaceuticalcomposition is an antihypertensive (e.g., labetalol, metoprolol,hydralazine, nitroglycerin, nicardipine, sodium nitroprusside,clevidipine), a diuretic (e.g., a thiazide diuretic, chlorthalidone,furosemide, hydrochlorothiazide, indapamide, metolazone, amiloridehydrochloride, spironolactone, triamterene) or aspirin. In someembodiments, the active agent of said at least one other pharmaceuticalcomposition is an angiotensin-converting enzyme (ACE) inhibitor (e.g.ramipril, ramiprilat, captopril, lisinopril) or an angiotensin IIreceptor blocker (e.g., losartan, olmesartan, valsartan). In someembodiments, the active agent of said at least one other pharmaceuticalcomposition is a vasodilator. In some embodiments, the active agent ofsaid at least one other pharmaceutical composition is an anticoagulant(e.g., acenocoumarol, phenprocoumon, warfarin heparin, low molecularweight heparin) or inhibitor of platelet aggregation (e.g., clopidogrel,ticlopidine, cilostazol, dipyridamole, eptifibatide, aspirin, abciximab,eptifibatide, tirofiban). In some embodiments, the active agent of saidat least one other pharmaceutical composition is a thrombolytic (e.g.,streptokinase, urokinase, alteplase, reteplase, tenecteplase). In someembodiments, the active agent of said at least one other pharmaceuticalcomposition is digitalis. In some embodiments, the active agent of saidat least one other pharmaceutical composition is digoxin or nesiritide.In some embodiments, the active agent of said at least one otherpharmaceutical composition is oxygen. In some embodiments, the activeagent of said at least one other pharmaceutical composition is athrombin inhibitor (e.g., hirudin, bivalirudin). In some embodiments,the active agent of said at least one other pharmaceutical compositionis a nitrate (e.g., glyceryl trinitrate (GTN)/nitroglycerin, isosorbidedinitrate, isosorbide mononitrate). In some embodiments, the activeagent of said at least one other pharmaceutical composition is ananalgesic (e.g., morphine sulfate). In some embodiments, the activeagent of said at least one other pharmaceutical composition is a renininhibitor. In some embodiments, the active agent of said at least oneother pharmaceutical composition is an endothelin A receptor inhibitor.In some embodiments, the active agent of said at least one otherpharmaceutical composition is an aldosterone inhibitor.

The present disclosure also provides uses of an anti-IL-1β bindingantibody or binding fragment thereof which has a lower IC₅₀ than anIL-1β receptor antagonist in a human whole blood IL-1β inhibition assaythat measures IL-1β induced production of IL-8, in the manufacture of acomposition for use in the reduction, prevention or treatment of acardiac event or a cardiovascular disease.

These IL-1β binding antibodies and binding fragments thereof, as well assuitable dose amounts and dosing regimens and/or other pharmaceuticalcompositions comprising an active agent other than an anti-IL-1βantibody or fragment thereof, as provided herein, may be used in or withany of the aforementioned methods and/or pharmaceutical compositions,including for example:

Methods and/or pharmaceutical compositions for use in reducing acardiovascular event (e.g., delaying time to event, reducing likelihoodor risk of event, preventing an event, reducing severity of event,reducing time to recovery) in a subject with a history of at least onerisk factor for cardiovascular disease, comprising administering to saidsubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof, and wherein the cardiovascularevent is myocardial infarction, stroke, cardiovascular death, congestiveheart failure, cardiac arrest, acute coronary syndrome, angina, or arevascularization procedure;

Methods and/or pharmaceutical compositions for use in reducing acardiovascular event (e.g., delaying time to event, reducing likelihoodor risk of event, preventing an event, reducing severity of event,reducing time to recovery) in a subject with a history of a previouscardiovascular event, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof, and wherein said cardiovascular event ismyocardial infarction, stroke, cardiovascular death, congestive heartfailure, cardiac arrest, acute coronary syndrome, angina or arevascularization procedure;

Methods and/or pharmaceutical compositions for use in reducing mortalityfollowing a cardiovascular event in a subject, comprising administeringto said subject a therapeutically effective amount of an anti-IL-1βbinding antibody or binding fragment thereof;

Methods and/or pharmaceutical compositions for use in reducing acardiovascular event in a subject with a history of at least one riskfactor for cardiovascular disease, comprising administering to saidsubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof, and wherein said risk factor isnot Type 2 diabetes, obesity, hyperglycemia, dyslipidemia,hyperlipidemia, chronic renal failure, high blood glucose, chronickidney disease, hypertension, atherosclerosis or metabolic syndrome;

Methods and/or pharmaceutical compositions for use in treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof and at least one other pharmaceuticalcomposition comprising an active agent other than an IL-1β antibody orfragment;

Methods and/or pharmaceutical compositions for use in treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof and a revascularization procedure;

Methods and/or pharmaceutical compositions for use in treatingcardiovascular disease, including, for example, acute cardiovasculardisease or chronic cardiovascular disease, in a subject, comprisingadministering to said subject a therapeutically effective amount of ananti-IL-1β binding antibody or binding fragment thereof and arevascularization procedure;

Methods and/or pharmaceutical compositions for use in reducingrestenosis in a subject following a revascularization procedure,comprising administering to said subject a therapeutically effectiveamount of an anti-IL-1β binding antibody or binding fragment thereof;

Methods and/or pharmaceutical compositions for use in treating acutehypertension in a subject, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof and one or more antihypertensive agents;

Methods and/or pharmaceutical compositions for use in reducing,preventing or treating a cardiovascular event or disease (e.g., acutecardiovascular disease or chronic cardiovascular disease) in a subject,comprising administering to the subject an anti-IL-1β binding antibodyor binding fragment thereof in combination with a medical or surgicalintervention;

Methods and/or pharmaceutical compositions for use in inhibitingplatelet activity in a subject, comprising administering to said subjecta therapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof;

Methods and/or pharmaceutical compositions for use in reducing acardiovascular event in a subject with a history of at least one riskfactor for cardiovascular disease, comprising (a) identifying,diagnosing or selecting the subject with the history of at least onerisk factor for cardiovascular disease and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof, and wherein the cardiovascularevent is myocardial infarction, stroke, cardiovascular death, congestiveheart failure, cardiac arrest, acute coronary syndrome, angina, or arevascularization procedure;

Methods and/or pharmaceutical compositions for use in reducing acardiovascular event in a subject with a history of a previouscardiovascular event, comprising (a) identifying, diagnosing orselecting the subject with the history of the previous cardiovascularevent and (b) administering to the subject a therapeutically effectiveamount of an anti-IL-1β binding antibody or binding fragment thereof,and wherein the cardiovascular event is myocardial infarction, stroke,acute coronary syndrome, angina or a revascularization procedure;

Methods and/or pharmaceutical compositions for use in reducing mortalityfollowing a cardiovascular event in a subject, comprising (a)identifying, diagnosing or selecting the subject having thecardiovascular event and (b) administering to the subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof;

Methods and/or pharmaceutical compositions for use in reducing acardiovascular event in a subject with a history of at least one riskfactor for cardiovascular disease, comprising (a) identifying,diagnosing or selecting the subject with the history of at least onerisk factor for cardiovascular disease and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof, and wherein the risk factor is notType 2 diabetes, obesity, hyperglycemia, dyslipidemia, hyperlipidemia,chronic renal failure, high blood glucose, chronic kidney disease,hypertension, atherosclerosis or metabolic syndrome;

Methods and/or pharmaceutical compositions for use in treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising (a) identifying, diagnosing or selectingthe subject with the cardiovascular event and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof and at least one otherpharmaceutical composition comprising an active agent other than anIL-1β antibody or fragment;

Methods and/or pharmaceutical compositions for use in treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising (a) identifying, diagnosing or selectingthe subject with the cardiovascular event and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof and (e.g., in conjunction with) arevascularization procedure;

Methods and/or pharmaceutical compositions for use in reducingrestenosis in a subject following a revascularization procedure,comprising (a) identifying, diagnosing or selecting the subject with therevascularization procedure and (b) administering to the subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof;

Methods and/or pharmaceutical compositions for use in treating acutehypertension in a subject comprising (a) identifying, diagnosing orselecting the subject with acute hypertension and (b) administering tothe subject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof and one or more antihypertensiveagents.

It should be understood that where the present specification providesmethods of using IL-1β antibodies or binding fragments thereof withcertain properties (such as Kd values or IC₅₀ values), such as forexample, for the reduction, prevention or treatment of cardiovascularevents and/or cardiovascular diseases, including acute cardiovasculardisease or chronic cardiovascular disease, this also means to embody theuse of such antibodies or fragments thereof in the manufacture of amedicament for use in these methods. Further, the disclosure alsoencompasses IL-1β antibodies or binding fragments thereof having theseproperties as well as pharmaceutical compositions comprising theseantibodies or fragments thereof for use in the methods provided herein,such as for example, for the reduction, prevention or treatment ofcardiovascular events and/or cardiovascular diseases, including acutecardiovascular disease or chronic cardiovascular disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing serum concentrations following IVadministration of 0.01, 0.03, 0.1, 0.3, or 1.0 mg/kg of an anti-IL-1βantibody in human subjects.

FIG. 2 is a graph showing serum concentrations following SCadministration of 0.03, 0.1 and 0.3 mg/kg of an anti-IL-1β antibody inhuman subjects

FIG. 3 is a graph showing median percent change in CRP at day 28following administration of 0.01, 0.03, 0.1, 0.3, or 1.0 mg/kg of ananti-IL-1β antibody in human subjects.

FIG. 4 is graphs showing changes in echocardiographic values in amyocardial infarction animal model.

FIG. 5 is graphs showing measurements of akinetic segments (surrogatefor infarct size), anterior wall (infarct) thickness, MPI or Tei index(marker of combined systolic and diastolic dysfunction and a surrogatemarker for heart failure related mortality), and TAPSE (marker of rightventricular function and a surrogate marker for AMI related mortality ina myocardial infarction animal model.

FIG. 6 is graphs showing inhibition of the release of macrophage-inducedpro-inflammatory cytokines from endothelial cells.

FIG. 7 is graphs showing inhibition of the release of macrophage-inducedcytokines and degradative enzymes from smooth muscle cells.

FIG. 8 is graphs showing reduction in the formation of atheroscleroticlesions in the aortas of ApoE knockout mice.

FIG. 9 is photographs of en face analysis showing reduction in theformation of atherosclerotic lesions in the aortas of ApoE knockoutmice.

DETAILED DESCRIPTION

The present disclosure relates to methods and related articles ofmanufacture for the treatment and/or prevention of cardiovasculardisease, including, for example, acute cardiovascular disease or chroniccardiovascular disease. The methods may be used for reducing, treatingor preventing a cardiovascular event, such as myocardial infarction,stroke, cardiovascular death, congestive heart failure, cardiac arrest,acute coronary syndrome, angina, or a revascularization procedure in asubject, including in a subject with a history of a risk factor forcardiovascular disease. The methods may also be used to reduce mortalityfollowing a cardiovascular event in a subject. Use of anti-IL-1β bindingantibodies or binding fragments as disclosed herein, offers potentialadvantages over previously available options, such as for examplegreater safety (e.g., reduced side effects), greater efficacy, targetingof the inflammatory component of disease, and/or less frequent dosing.

The interleukin-1 (IL-1) family of cytokines has been implicated inseveral disease states such as rheumatoid arthritis (RA),osteoarthritis, Crohn's disease, ulcerative colitis (UC), septic shock,chronic obstructive pulmonary disease (COPD), asthma, graft versus hostdisease, atherosclerosis, adult T-cell leukemia, multiple myeloma,multiple sclerosis, stroke, and Alzheimer's disease. IL-1 family membersinclude IL-1α, IL-1β, and IL-1Ra. Although related by their ability tobind to IL-1 receptors (IL-1R1, IL-1R2), each of these cytokines isexpressed by a different gene and has a different primary amino acidsequence. Furthermore, the physiological activities of these cytokinescan be distinguished from each other.

Compounds that disrupt IL-1 receptor signaling have been investigated astherapeutic agents to treat IL-1 mediated diseases, such as for examplesome of the aforementioned diseases. These compounds include recombinantIL-1Ra (Amgen Inc., Thousand Oaks, Calif.), IL-1 receptor “trap” peptide(Regeneron Inc., Tarrytown, N.Y.), as well as animal-derived IL-1βantibodies and recombinant IL-1β antibodies and fragments thereof.Compounds that directly target the IL-1β ligand are believed to providea superior strategy, particularly when administering an IL-1β antibodywith high affinity.

Antibodies, Humanized Antibodies, and Human Engineered Antibodies

IL-1 (e.g., IL-1β) binding antibodies may be provided as polyclonalantibodies, monoclonal antibodies (mAbs), recombinant antibodies,chimeric antibodies, CDR-grafted antibodies, fully human antibodies,single chain antibodies, and/or bispecific antibodies, as well asfragments, including variants and derivatives thereof, provided by knowntechniques, including, but not limited to enzymatic cleavage, peptidesynthesis or recombinant techniques.

Antibodies generally comprise two heavy chain polypeptides and two lightchain polypeptides, though single domain antibodies having one heavychain and one light chain, and heavy chain antibodies devoid of lightchains are also contemplated. There are five types of heavy chains,called alpha, delta, epsilon, gamma and mu, based on the amino acidsequence of the heavy chain constant domain. These different types ofheavy chains give rise to five classes of antibodies, IgA (includingIgA₁ and IgA₂), IgD, IgE, IgG and IgM, respectively, including foursubclasses of IgG, namely IgG₁, IgG₂, IgG₃ and IgG₄. There are also twotypes of light chains, called kappa (κ) or lambda (λ) based on the aminoacid sequence of the constant domains. A full-length antibody includes aconstant domain and a variable domain. The constant region need not bepresent in an antigen binding fragment of an antibody. Antigen bindingfragments of an antibody disclosed herein can include Fab, Fab′,F(ab′)₂, and F(v) antibody fragments. As discussed in more detail below,IL-1β binding fragments encompass antibody fragments and antigen-bindingpolypeptides that will bind IL-1β.

Each of the heavy chain and light chain sequences of an antibody, orantigen binding fragment thereof, includes a variable region with threecomplementarity determining regions (CDRs) as well as non-CDR frameworkregions (FRs). The terms “heavy chain” and “light chain,” as usedherein, mean the heavy chain variable region and the light chainvariable region, respectively, unless otherwise noted. Heavy chain CDRsare referred to herein as CDR-H1, CDR-H2, and CDR-H3. Light chain CDRsare referred to herein as CDR-L1, CDR-L2, and CDR-L3. Variable regionsand CDRs in an antibody sequence can be identified (i) according togeneral rules that have been developed in the art or (ii) by aligningthe sequences against a database of known variable regions. Methods foridentifying these regions are described in Kontermann and Dubel, eds.,Antibody Engineering, Springer, New York, N.Y., 2001, and Dinarello etal., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken,N.J., 2000. Databases of antibody sequences are described in and can beaccessed through “The Kabatman” database at www.bioinf.org.uk/abs(maintained by A.C. Martin in the Department of Biochemistry & MolecularBiology University College London, London, England) and VBASE2 atwww.vbase2.org, as described in Retter et al., Nucl. Acids Res.,33(Database issue): D671-D674 (2005). The “Kabatman” database web sitealso includes general rules of thumb for identifying CDRs. The term“CDR,” as used herein, is as defined in Kabat et al., Sequences ofImmunological Interest, 5^(th) ed., U.S. Department of Health and HumanServices, 1991, unless otherwise indicated.

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) immunizing injections of therelevant antigen and an adjuvant, using standard techniques known in theart. An improved antibody response may be obtained by conjugating therelevant antigen to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride or other agentsknown in the art.

Monoclonal antibody refers to an antibody obtained from a population ofsubstantially homogeneous antibodies. Monoclonal antibodies aregenerally highly specific, and may be directed against a singleantigenic site, in contrast to conventional (polyclonal) antibodypreparations that typically include different antibodies directedagainst different determinants (epitopes). In addition to theirspecificity, the monoclonal antibodies are advantageous in that they aresynthesized by the homogeneous culture, uncontaminated by otherimmunoglobulins with different specificities and characteristics.

Monoclonal antibodies to be used in accordance with the presentdisclosure may be made by the hybridoma method first described by Kohleret al., (Nature, 256:495-7, 1975), or may be made by recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodiesmay also be isolated from phage antibody libraries using the techniquesdescribed in, for example, Clackson et al., (Nature 352:624-628, 1991)and Marks et al., (J. Mol. Biol. 222:581-597, 1991).

It is further contemplated that antibodies of the present disclosure maybe used as smaller antigen binding fragments of the antibody well-knownin the art and described herein. The present disclosure encompasses IL-1(e.g., IL-1β) binding antibodies that include two full length heavychains and two full length light chains. Alternatively, the IL-1βbinding antibodies can be constructs such as single chain antibodies or“mini” antibodies that retain binding activity to IL-1β. Such constructscan be prepared by methods known in the art such as, for example, thePCR mediated cloning and assembly of single chain antibodies forexpression in E. coli (as described in Antibody Engineering, Thepractical approach series, J. McCafferty, H. R. Hoogenboom, and D. J.Chiswell, editors, Oxford University Press, 1996). In this type ofconstruct, the variable portions of the heavy and light chains of anantibody molecule are PCR amplified from cDNA. The resulting ampliconsare then assembled, for example, in a second PCR step, through a linkerDNA that encodes a flexible protein linker composed of the amino acidsGly and Ser. This linker allows the variable heavy and light chainportions to fold in such a way that the antigen binding pocket isregenerated and antigen is bound with affinities often comparable to theparent full-length dimeric immunoglobulin molecule.

The IL-1 (e.g., IL-1β) binding antibodies and fragments encompassvariants of the exemplary antibodies, fragments and sequences disclosedherein. Variants include peptides and polypeptides comprising one ormore amino acid sequence substitutions, deletions, and/or additions thathave the same or substantially the same affinity and specificity ofepitope binding as one or more of the exemplary antibodies, fragmentsand sequences disclosed herein. Thus, variants include peptides andpolypeptides comprising one or more amino acid sequence substitutions,deletions, and/or additions to the exemplary antibodies, fragments andsequences disclosed herein where such substitutions, deletions and/oradditions do not cause substantial changes in affinity and specificityof epitope binding. For example, a variant of an antibody or fragmentmay result from one or more changes to an antibody or fragment, wherethe changed antibody or fragment has the same or substantially the sameaffinity and specificity of epitope binding as the starting sequence.Variants may be naturally occurring, such as allelic or splice variants,or may be artificially constructed. Variants may be prepared from thecorresponding nucleic acid molecules encoding said variants. Variants ofthe present antibodies and IL-1β binding fragments may have changes inlight and/or heavy chain amino acid sequences that are naturallyoccurring or are introduced by in vitro engineering of native sequencesusing recombinant DNA techniques. Naturally occurring variants include“somatic” variants which are generated in vivo in the corresponding germline nucleotide sequences during the generation of an antibody responseto a foreign antigen.

Variants of IL-1 (e.g., IL-1β) binding antibodies and binding fragmentsmay also be prepared by mutagenesis techniques. For example, amino acidchanges may be introduced at random throughout an antibody coding regionand the resulting variants may be screened for binding affinity forIL-1β or for another property. Alternatively, amino acid changes may beintroduced in selected regions of an IL-1β antibody, such as in thelight and/or heavy chain CDRs, and/or in the framework regions, and theresulting antibodies may be screened for binding to IL-1β or some otheractivity. Amino acid changes encompass one or more amino acidsubstitutions in a CDR, ranging from a single amino acid difference tothe introduction of multiple permutations of amino acids within a givenCDR, such as CDR3. In another method, the contribution of each residuewithin a CDR to IL-1β binding may be assessed by substituting at leastone residue within the CDR with alanine. Lewis et al. (1995), Mol.Immunol. 32: 1065-72. Residues which are not optimal for binding toIL-1β may then be changed in order to determine a more optimum sequence.Also encompassed are variants generated by insertion of amino acids toincrease the size of a CDR, such as CDR3. For example, most light chainCDR3 sequences are nine amino acids in length. Light chain sequences inan antibody which are shorter than nine residues may be optimized forbinding to IL-1β by insertion of appropriate amino acids to increase thelength of the CDR.

Variants may also be prepared by “chain shuffling” of light or heavychains. Marks et al. (1992), Biotechnology 10: 779-83. A single light(or heavy) chain can be combined with a library having a repertoire ofheavy (or light) chains and the resulting population is screened for adesired activity, such as binding to IL-1β. This permits screening of agreater sample of different heavy (or light) chains in combination witha single light (or heavy) chain than is possible with librariescomprising repertoires of both heavy and light chains.

The IL-1 (e.g., IL-1β) binding antibodies and fragments of the presentdisclosure encompass derivatives of the exemplary antibodies, fragmentsand sequences disclosed herein. Derivatives include polypeptides orpeptides, or variants, fragments or derivatives thereof, which have beenchemically modified. Examples include covalent attachment of one or morepolymers, such as water soluble polymers, N-linked, or O-linkedcarbohydrates, sugars, phosphates, and/or other such molecules. Thederivatives are modified in a manner that is different from naturallyoccurring or starting peptide or polypeptides, either in the type orlocation of the molecules attached. Derivatives further include deletionof one or more chemical groups which are naturally present on thepeptide or polypeptide.

The IL-1β binding antibodies and fragments can be bispecific. Bispecificantibodies or fragments can be of several configurations. For example,bispecific antibodies may resemble single antibodies (or antibodyfragments) but have two different antigen binding sites (variableregions). Bispecific antibodies can be produced by chemical techniques(Kranz et al. (1981), Proc. Natl. Acad. Sci. USA, 78: 5807), by“polydoma” techniques (U.S. Pat. No. 4,474,893) or by recombinant DNAtechniques. Bispecific antibodies of the present disclosure can havebinding specificities for at least two different epitopes, at least oneof which is an epitope of IL-1β. The IL-1β binding antibodies andfragments can also be heteroantibodies. Heteroantibodies are two or moreantibodies, or antibody binding fragments (Fab) linked together, eachantibody or fragment having a different specificity.

Techniques for creating recombinant DNA versions of the antigen-bindingregions of antibody molecules which bypass the generation of monoclonalantibodies are contemplated for the present IL-1 (e.g., IL-1β) bindingantibodies and fragments. DNA is cloned into a bacterial expressionsystem. One example of such a technique suitable for the practice of thepresent disclosure uses a bacteriophage lambda vector system having aleader sequence that causes the expressed Fab protein to migrate to theperiplasmic space (between the bacterial cell membrane and the cellwall) or to be secreted. One can rapidly generate and screen greatnumbers of functional Fab fragments for those which bind IL-1β. SuchIL-1β binding agents (Fab fragments with specificity for an IL-1βpolypeptide) are specifically encompassed within the IL-1β bindingantibodies and fragments of the present disclosure.

The present IL-1 (e.g., IL-1β) binding antibodies and fragments can behumanized or human engineered antibodies. As used herein, a humanizedantibody, or antigen binding fragment thereof, is a recombinantpolypeptide that comprises a portion of an antigen binding site from anon-human antibody and a portion of the framework and/or constantregions of a human antibody. A human engineered antibody or antibodyfragment is a non-human (e.g., mouse) antibody that has been engineeredby modifying (e.g., deleting, inserting, or substituting) amino acids atspecific positions so as to reduce or eliminate any detectableimmunogenicity of the modified antibody in a human.

Humanized antibodies include chimeric antibodies and CDR-graftedantibodies. Chimeric antibodies are antibodies that include a non-humanantibody variable region linked to a human constant region. Thus, inchimeric antibodies, the variable region is mostly non-human, and theconstant region is human. Chimeric antibodies and methods for makingthem are described in Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6841-6855 (1984), Boulianne, et al., Nature, 312: 643-646 (1984), andPCT Application Publication WO 86/01533. Although, they can be lessimmunogenic than a mouse monoclonal antibody, administrations ofchimeric antibodies have been associated with human anti-mouse antibodyresponses (HAMA) to the non-human portion of the antibodies. Chimericantibodies can also be produced by splicing the genes from a mouseantibody molecule of appropriate antigen-binding specificity togetherwith genes from a human antibody molecule of appropriate biologicalactivity, such as the ability to activate human complement and mediateADCC. Morrison et al. (1984), Proc. Natl. Acad. Sci., 81: 6851;Neuberger et al. (1984), Nature, 312: 604. One example is thereplacement of a Fc region with that of a different isotype.

CDR-grafted antibodies are antibodies that include the CDRs from anon-human “donor” antibody linked to the framework region from a human“recipient” antibody. Generally, CDR-grafted antibodies include morehuman antibody sequences than chimeric antibodies because they includeboth constant region sequences and variable region (framework) sequencesfrom human antibodies. Thus, for example, a CDR-grafted humanizedantibody of the present disclosure can comprise a heavy chain thatcomprises a contiguous amino acid sequence (e.g., about 5 or more, 10 ormore, or even 15 or more contiguous amino acid residues) from theframework region of a human antibody (e.g., FR-1, FR-2, or FR-3 of ahuman antibody) or, optionally, most or all of the entire frameworkregion of a human antibody. CDR-grafted antibodies and methods formaking them are described in, Jones et al., Nature, 321: 522-525 (1986),Riechmann et al., Nature, 332: 323-327 (1988), and Verhoeyen et al.,Science, 239: 1534-1536 (1988)). Methods that can be used to producehumanized antibodies also are described in U.S. Pat. Nos. 4,816,567,5,721,367, 5,837,243, and 6,180,377. CDR-grafted antibodies areconsidered less likely than chimeric antibodies to induce an immunereaction against non-human antibody portions. However, it has beenreported that framework sequences from the donor antibodies are requiredfor the binding affinity and/or specificity of the donor antibody,presumably because these framework sequences affect the folding of theantigen-binding portion of the donor antibody. Therefore, when donor,non-human CDR sequences are grafted onto unaltered human frameworksequences, the resulting CDR-grafted antibody can exhibit, in somecases, loss of binding avidity relative to the original non-human donorantibody. See, e.g., Riechmann et al., Nature, 332: 323-327 (1988), andVerhoeyen et al., Science, 239: 1534-1536 (1988).

Human engineered antibodies include for example “veneered” antibodiesand antibodies prepared using HUMAN ENGINEERING™ technology (U.S. Pat.No. 5,869,619). HUMAN ENGINEERING™ technology is commercially available,and involves altering an non-human antibody or antibody fragment, suchas a mouse or chimeric antibody or antibody fragment, by making specificchanges to the amino acid sequence of the antibody so as to produce amodified antibody with reduced immunogenicity in a human thatnonetheless retains the desirable binding properties of the originalnon-human antibodies. Generally, the technique involves classifyingamino acid residues of a non-human (e.g., mouse) antibody as “low risk”,“moderate risk”, or “high risk” residues. The classification isperformed using a global risk/reward calculation that evaluates thepredicted benefits of making particular substitution (e.g., forimmunogenicity in humans) against the risk that the substitution willaffect the resulting antibody's folding and/or antigen-bindingproperties. Thus, a low risk position is one for which a substitution ispredicted to be beneficial because it is predicted to reduceimmunogenicity without significantly affecting antigen bindingproperties. A moderate risk position is one for which a substitution ispredicted to reduce immunogenicity, but is more likely to affect proteinfolding and/or antigen binding. High risk positions contain residuesmost likely to be involved in proper folding or antigen binding.Generally, low risk positions in a non-human antibody are substitutedwith human residues, high risk positions are rarely substituted, andhumanizing substitutions at moderate risk positions are sometimes made,although not indiscriminately. Positions with prolines in the non-humanantibody variable region sequence are usually classified as at leastmoderate risk positions.

The particular human amino acid residue to be substituted at a given lowor moderate risk position of a non-human (e.g., mouse) antibody sequencecan be selected by aligning an amino acid sequence from the non-humanantibody's variable regions with the corresponding region of a specificor consensus human antibody sequence. The amino acid residues at low ormoderate risk positions in the non-human sequence can be substituted forthe corresponding residues in the human antibody sequence according tothe alignment. Techniques for making human engineered proteins aredescribed in greater detail in Studnicka et al., Protein Engineering, 7:805-814 (1994), U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and5,869,619, and PCT Application Publication WO 93/11794.

“Veneered” antibodies are non-human or humanized (e.g., chimeric orCDR-grafted antibodies) antibodies that have been engineered to replacecertain solvent-exposed amino acid residues so as to further reducetheir immunogenicity or enhance their function. As surface residues of achimeric antibody are presumed to be less likely to affect properantibody folding and more likely to elicit an immune reaction, veneeringof a chimeric antibody can include, for instance, identifyingsolvent-exposed residues in the non-human framework region of a chimericantibody and replacing at least one of them with the correspondingsurface residues from a human framework region. Veneering can beaccomplished by any suitable engineering technique, including the use ofthe above-described HUMAN ENGINEERING™ technology.

In a different approach, a recovery of binding avidity can be achievedby “de-humanizing” a CDR-grafted antibody. De-humanizing can includerestoring residues from the donor antibody's framework regions to theCDR grafted antibody, thereby restoring proper folding. Similar“de-humanization” can be achieved by (i) including portions of the“donor” framework region in the “recipient” antibody or (ii) graftingportions of the “donor” antibody framework region into the recipientantibody (along with the grafted donor CDRs).

For a further discussion of antibodies, humanized antibodies, humanengineered, and methods for their preparation, see Kontermann and Dubel,eds., Antibody Engineering, Springer, New York, N.Y., 2001.

Exemplary humanized or human engineered antibodies include IgG, IgM,IgE, IgA, and IgD antibodies. The present antibodies can be of any class(IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa orlambda light chain. For example, a human antibody can comprise an IgGheavy chain or defined fragment, such as at least one of isotypes, IgG1,IgG2, IgG3 or IgG4. As a further example, the present antibodies orfragments can comprise an IgG1 heavy chain and an IgG1 light chain.

The present antibodies and fragments can be human antibodies, such asantibodies which bind IL-1β polypeptides and are encoded by nucleic acidsequences which are naturally occurring somatic variants of humangermline immunoglobulin nucleic acid sequence, and fragments, syntheticvariants, derivatives and fusions thereof. Such antibodies may beproduced by any method known in the art, such as through the use oftransgenic mammals (such as transgenic mice) in which the nativeimmunoglobulin repertoire has been replaced with human V-genes in themammal chromosome. Such mammals appear to carry out VDJ recombinationand somatic hypermutation of the human germline antibody genes in anormal fashion, thus producing high affinity antibodies with completelyhuman sequences.

Human antibodies to target protein can also be produced using transgenicanimals that have no endogenous immunoglobulin production and areengineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals having a human Ig locus whereinthe animals do not produce functional endogenous immunoglobulins due tothe inactivation of endogenous heavy and light chain loci. WO 91/00906also discloses transgenic non-primate mammalian hosts capable ofmounting an immune response to an immunogen, wherein the antibodies haveprimate constant and/or variable regions, and wherein the endogenousimmunoglobulin encoding loci are substituted or inactivated. WO 96/30498and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system tomodify the immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. WO 94/02602 discloses non-human mammalian hosts havinginactivated endogenous Ig loci and functional human Ig loci. U.S. Pat.No. 5,939,598 discloses methods of making transgenic mice in which themice lack endogenous heavy chains, and express an exogenousimmunoglobulin locus comprising one or more xenogeneic constant regions.See also, U.S. Pat. Nos. 6,114,598 6,657,103 and 6,833,268.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. Thispublication discloses monoclonal antibodies against a variety ofantigenic molecules including IL-6, IL-8, TNFa, human CD4, L selectin,gp39, and tetanus toxin. The monoclonal antibodies can be tested for theability to inhibit or neutralize the biological activity orphysiological effect of the corresponding protein. WO 96/33735 disclosesthat monoclonal antibodies against IL-8, derived from immune cells oftransgenic mice immunized with IL-8, blocked IL-8 induced functions ofneutrophils. Human monoclonal antibodies with specificity for theantigen used to immunize transgenic animals are also disclosed in WO96/34096 and U.S. patent application no. 20030194404; and U.S. patentapplication no. 20030031667.

Additional transgenic animals useful to make monoclonal antibodiesinclude the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429and Fishwild, et al. (Nat. Biotechnol. 14:845-851, 1996), which containsgene sequences from unrearranged human antibody genes that code for theheavy and light chains of human antibodies. Immunization of aHuMAb-MOUSE® enables the production of fully human monoclonal antibodiesto the target protein.

Also, Ishida et al. (Cloning Stem Cells. 4:91-102, 2002) describes theTransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments ofhuman DNA and which incorporates the entire human immunoglobulin (hIg)loci. The TCMOUSE™ has a fully diverse repertoire of hIgs, including allthe subclasses of IgGs (IgG1-G4). Immunization of the TC MOUSE™ withvarious human antigens produces antibody responses comprising humanantibodies.

See also Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); and U.S. Pat. No. 5,591,669, U.S. Pat. No.5,589,369, U.S. Pat. No. 5,545,807; and U.S Patent Publication No.20020199213. U.S. Patent Publication No. 20030092125 describes methodsfor biasing the immune response of an animal to the desired epitope.Human antibodies may also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human antibodies can also be generated through the in vitro screening ofantibody display libraries. See Hoogenboom et al. (1991), J. Mol. Biol.227: 381; and Marks et al. (1991), J. Mol. Biol. 222: 581. Variousantibody-containing phage display libraries have been described and maybe readily prepared. Libraries may contain a diversity of human antibodysequences, such as human Fab, Fv, and scFv fragments, that may bescreened against an appropriate target. Phage display libraries maycomprise peptides or proteins other than antibodies which may bescreened to identify selective binding agents of IL-1β.

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a means formaking human antibodies directly. The antibodies produced by phagetechnology are produced as antigen binding fragments-usually Fv or Fabfragments-in bacteria and thus lack effector functions. Effectorfunctions can be introduced by one of two strategies: The fragments canbe engineered either into complete antibodies for expression inmammalian cells, or into bispecific antibody fragments with a secondbinding site capable of triggering an effector function.

The present disclosure contemplates a method for producingtarget-specific antibody or antigen-binding portion thereof comprisingthe steps of synthesizing a library of human antibodies on phage,screening the library with target protein or a portion thereof,isolating phage that bind target, and obtaining the antibody from thephage. By way of example, one method for preparing the library ofantibodies for use in phage display techniques comprises the steps ofimmunizing a non-human animal comprising human immunoglobulin loci withtarget antigen or an antigenic portion thereof to create an immuneresponse, extracting antibody producing cells from the immunized animal;isolating RNA from the extracted cells, reverse transcribing the RNA toproduce cDNA, amplifying the cDNA using a primer, and inserting the cDNAinto a phage display vector such that antibodies are expressed on thephage. Recombinant target-specific antibodies of the present disclosuremay be obtained in this way.

Phage-display processes mimic immune selection through the display ofantibody repertoires on the surface of filamentous bacteriophage, andsubsequent selection of phage by their binding to an antigen of choice.One such technique is described in WO 99/10494, which describes theisolation of high affinity and functional agonistic antibodies for MPLand msk receptors using such an approach. Antibodies of the presentdisclosure can be isolated by screening of a recombinant combinatorialantibody library, preferably a scFv phage display library, preparedusing human V_(L) and V_(H) cDNAs prepared from mRNA derived from humanlymphocytes. Methodologies for preparing and screening such librariesare known in the art. See e.g., U.S. Pat. No. 5,969,108. There arecommercially available kits for generating phage display libraries(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no.240612). There are also other methods and reagents that can be used ingenerating and screening antibody display libraries (see, e.g., Ladneret al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al.PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffertyet al. PCT Publication No. WO 92/01047; Garrard et al. PCT PublicationNo. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982.

In one embodiment, to isolate human antibodies specific for the targetantigen with the desired characteristics, a human V_(H) and V_(L),library are screened to select for antibody fragments having the desiredspecificity. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described herein and in the art(McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al.,(Nature 348:552-554, 1990); and Griffiths et al., (EMBO J. 12:725-734,1993). The scFv antibody libraries preferably are screened using targetprotein as the antigen.

Alternatively, the Fd fragment (V_(H)-C_(H)1) and light chain(V_(L)-C_(L)) of antibodies are separately cloned by PCR and recombinedrandomly in combinatorial phage display libraries, which can then beselected for binding to a particular antigen. The Fab fragments areexpressed on the phage surface, i.e., physically linked to the genesthat encode them. Thus, selection of Fab by antigen binding co-selectsfor the Fab encoding sequences, which can be amplified subsequently.Through several rounds of antigen binding and re-amplification, aprocedure termed panning, Fab specific for the antigen are enriched andfinally isolated.

In 1994, an approach for the humanization of antibodies, called “guidedselection”, was described. Guided selection utilizes the power of thephage display technique for the humanization of mouse monoclonalantibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903(1994)). For this, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library, and theresulting hybrid Fab library may then be selected with antigen. Themouse Fd fragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol,222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson,T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, invitro selection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); Winter, G., et al., Annu. Rev.Immunol. 12, 433-455 (1994); U.S. patent publication no. 20020004215 andWO 92/01047; U.S. patent publication no. 20030190317; and U.S. Pat. Nos.6,054,287 and 5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by CaptureLift,” Methods in Molecular Biology, Antibody Phage Display: Methods andProtocols 178: 187-193 (2002), and U.S. patent publication no.20030044772, published Mar. 6, 2003, describe methods for screeningphage-expressed antibody libraries or other binding molecules by capturelift, a method involving immobilization of the candidate bindingmolecules on a solid support.

Fv fragments are displayed on the surface of phage, by the associationof one chain expressed as a phage protein fusion (e.g., with M13 geneIII) with the complementary chain expressed as a soluble fragment. It iscontemplated that the phage may be a filamentous phage such as one ofthe class I phages: fd, M13, f1, If1, lke, ZJ/Z, Ff and one of the classII phages Xf, Pf1 and Pf3. The phage may be M13, or fd or a derivativethereof.

Once initial human V_(L) and V_(H) segments are selected, “mix andmatch” experiments, in which different pairs of the initially selectedV_(L) and V_(H) segments are screened for target binding, are performedto select preferred V_(L)/V_(H) pair combinations. Additionally, tofurther improve the quality of the antibody, the V_(L) and V_(H)segments of the preferred V_(L)/V_(H) pair(s) can be randomly mutated,preferably within the any of the CDR1, CDR2 or CDR3 region of V_(H)and/or V_(L), in a process analogous to the in vivo somatic mutationprocess responsible for affinity maturation of antibodies during anatural immune response. This in vitro affinity maturation can beaccomplished by amplifying V_(L) and V_(H) regions using PCR primerscomplimentary to the V_(H) CDR1, CDR2, and CDR3, or V_(L) CDR1, CDR2,and CDR3, respectively, which primers have been “spiked” with a randommixture of the four nucleotide bases at certain positions such that theresultant PCR products encode V_(L) and V_(H) segments into which randommutations have been introduced into the V_(H) and/or V_(L) CDR3 regions.These randomly mutated V_(L) and V_(H) segments can be rescreened forbinding to target antigen.

Following screening and isolation of an target specific antibody from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (e.g., fromthe phage genome) and subcloned into other expression vectors bystandard recombinant DNA techniques. If desired, the nucleic acid can befurther manipulated to create other antibody forms of the presentdisclosure, as described below. To express a recombinant human antibodyisolated by screening of a combinatorial library, the DNA encoding theantibody is cloned into a recombinant expression vector and introducedinto a mammalian host cell, as described herein.

It is contemplated that the phage display method may be carried out in amutator strain of bacteria or host cell. A mutator strain is a host cellwhich has a genetic defect which causes DNA replicated within it to bemutated with respect to its parent DNA. Example mutator strains areNR9046mutD5 and NR9046 mut T1.

It is also contemplated that the phage display method may be carried outusing a helper phage. This is a phage which is used to infect cellscontaining a defective phage genome and which functions to complementthe defect. The defective phage genome can be a phagemid or a phage withsome function encoding gene sequences removed. Examples of helper phagesare M13K07, M13K07 gene III no. 3; and phage displaying or encoding abinding molecule fused to a capsid protein.

Antibodies are also generated via phage display screening methods usingthe hierarchical dual combinatorial approach as disclosed in WO 92/01047in which an individual colony containing either an H or L chain clone isused to infect a complete library of clones encoding the other chain (Lor H) and the resulting two-chain specific binding member is selected inaccordance with phage display techniques such as those describedtherein. This technique is also disclosed in Marks et al,(Bio/Technology, 10:779-783, 1992).

Methods for display of peptides on the surface of yeast and microbialcells have also been used to identify antigen specific antibodies. See,for example, U.S. Pat. No. 6,699,658. Antibody libraries may be attachedto yeast proteins, such as agglutinin, effectively mimicking the cellsurface display of antibodies by B cells in the immune system.

In addition to phage display methods, antibodies may be isolated usingribosome mRNA display methods and microbial cell display methods.Selection of polypeptide using ribosome display is described in Hanes etal., (Proc. Natl. Acad Sci USA, 94:4937-4942, 1997) and U.S. Pat. Nos.5,643,768 and 5,658,754 issued to Kawasaki. Ribosome display is alsouseful for rapid large scale mutational analysis of antibodies. Theselective mutagenesis approach also provides a method of producingantibodies with improved activities that can be selected using ribosomaldisplay techniques.

The IL-1 (e.g., IL-1β) binding antibodies and fragments may comprise oneor more portions that do not bind IL-1β but instead are responsible forother functions, such as circulating half-life, direct cytotoxic effect,detectable labeling, or activation of the recipient's endogenouscomplement cascade or endogenous cellular cytotoxicity. The antibodiesor fragments may comprise all or a portion of the constant region andmay be of any isotype, including IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG(e.g. IgG1, IgG2, IgG3 or IgG4), or IgM. In addition to, or instead of,comprising a constant region, antigen-binding compounds of the presentdisclosure may include an epitope tag, a salvage receptor epitope, alabel moiety for diagnostic or purification purposes, or a cytotoxicmoiety such as a radionuclide or toxin.

The constant region (when present) of the present antibodies andfragments may be of the γ1, γ2, γ3, γ4, μ, β2, or δ or ε type,preferably of the y type, more preferably of the y, type, whereas theconstant part of a human light chain may be of the κ or λ type (whichincludes the λ₁, λ₂ and λ₃ subtypes) but is preferably of the κ type.

Variants also include antibodies or fragments comprising a modified Fcregion, wherein the modified Fc region comprises at least one amino acidmodification relative to a wild-type Fc region. The variant Fc regionmay be designed, relative to a comparable molecule comprising thewild-type Fc region, so as to bind Fc receptors with a greater or lesseraffinity.

For example, the present IL-1β binding antibodies and fragments maycomprise a modified Fc region. Fc region refers to naturally-occurringor synthetic polypeptides homologous to the IgG C-terminal domain thatis produced upon papain digestion of IgG. IgG Fc has a molecular weightof approximately 50 kD. In the present antibodies and fragments, anentire Fc region can be used, or only a half-life enhancing portion. Inaddition, many modifications in amino acid sequence are acceptable, asnative activity is not in all cases necessary or desired.

The Fc region can be mutated, if desired, to inhibit its ability to fixcomplement and bind the Fc receptor with high affinity. For murine IgGFc, substitution of Ala residues for Glu 318, Lys 320, and Lys 322renders the protein unable to direct ADCC. Substitution of Glu for Leu235 inhibits the ability of the protein to bind the Fc receptor withhigh affinity. Various mutations for human IgG also are known (see,e.g., Morrison et al., 1994, The Immunologist 2: 119 124 and Brekke etal., 1994, The Immunologist 2: 125).

In some embodiments, the present an antibodies or fragments are providedwith a modified Fc region where a naturally-occurring Fc region ismodified to increase the half-life of the antibody or fragment in abiological environment, for example, the serum half-life or a half-lifemeasured by an in vitro assay. Methods for altering the original form ofa Fc region of an IgG also are described in U.S. Pat. No. 6,998,253.

In certain embodiments, it may be desirable to modify the antibody orfragment in order to increase its serum half-life, for example, addingmolecules such as PEG or other water soluble polymers, includingpolysaccharide polymers, to antibody fragments to increase thehalf-life. This may also be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis) (see, International Publication No. WO96/32478).Salvage receptor binding epitope refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule.

A salvage receptor binding epitope can include a region wherein any oneor more amino acid residues from one or two loops of a Fc domain aretransferred to an analogous position of the antibody fragment. Even morepreferably, three or more residues from one or two loops of the Fcdomain are transferred. Still more preferred, the epitope is taken fromthe CH2 domain of the Fc region (e.g., of an IgG) and transferred to theCH₁, CH₃, or V_(H) region, or more than one such region, of theantibody. Alternatively, the epitope is taken from the CH2 domain of theFc region and transferred to the C_(L) region or V_(L) region, or both,of the antibody fragment. See also International applications WO97/34631 and WO 96/32478 which describe Fc variants and theirinteraction with the salvage receptor.

Mutation of residues within Fc receptor binding sites can result inaltered effector function, such as altered ADCC or CDC activity, oraltered half-life. Potential mutations include insertion, deletion orsubstitution of one or more residues, including substitution withalanine, a conservative substitution, a non-conservative substitution,or replacement with a corresponding amino acid residue at the sameposition from a different IgG subclass (e.g. replacing an IgG1 residuewith a corresponding IgG2 residue at that position). For example it hasbeen reported that mutating the serine at amino acid position 241 inIgG4 to proline (found at that position in IgG1 and IgG2) led to theproduction of a homogeneous antibody, as well as extending serumhalf-life and improving tissue distribution compared to the originalchimeric IgG4. (Angal et al., Mol. Immunol. 30:105-8, 1993).

Antibody fragments are portions of an intact full length antibody, suchas an antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); multispecific antibody fragments such as bispecific,trispecific, and multispecific antibodies (e.g., diabodies, triabodies,tetrabodies); minibodies; chelating recombinant antibodies; tribodies orbibodies; intrabodies; nanobodies; small modular immunopharmaceuticals(SMIP), adnectins, binding-domain immunoglobulin fusion proteins;camelized antibodies; V_(HH) containing antibodies; and any otherpolypeptides formed from antibody fragments.

The present disclosure includes IL-1β binding antibody fragmentscomprising any of the foregoing heavy or light chain sequences and whichbind IL-1β. The term fragments as used herein refers to any 3 or morecontiguous amino acids (e.g., 4 or more, 5 or more 6 or more, 8 or more,or even 10 or more contiguous amino acids) of the antibody andencompasses Fab, Fab′, F(ab′)₂, and F(v) fragments, or the individuallight or heavy chain variable regions or portion thereof. IL-1β bindingfragments include, for example, Fab, Fab′, F(ab′)₂, Fv and scFv. Thesefragments lack the Fc fragment of an intact antibody, clear more rapidlyfrom the circulation, and can have less non-specific tissue binding thanan intact antibody. See Wahl et al. (1983), J. Nucl. Med., 24: 316-25.These fragments can be produced from intact antibodies using well knownmethods, for example by proteolytic cleavage with enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab′)₂ fragments).

In vitro and cell based assays are well described in the art for use indetermining binding of IL-1β to IL-1 receptor type I (IL-1R1), includingassays that determining in the presence of molecules (such asantibodies, antagonists, or other inhibitors) that bind to IL-1β orIL-1R1. (see for example Evans et al., (1995), J. Biol. Chem.270:11477-11483; Vigers et al., (2000), J. Biol. Chem. 275:36927-36933;Yanofsky et al., (1996), Proc. Natl. Acad. Sci. USA 93:7381-7386;Fredericks et al., (2004), Protein

Eng. Des. Sel. 17:95-106; Slack et al., (1993), J. Biol. Chem.268:2513-2524; Smith et al., (2003), Immunity 18:87-96; Vigers et al.,(1997), Nature 386:190-194; Ruggiero et al., (1997), J. Immunol.158:3881-3887; Guo et al., (1995), J. Biol. Chem. 270:27562-27568;Svenson et al., (1995), Eur. J. Immunol. 25:2842-2850; Arend et al.,(1994), J. Immunol. 153:4766-4774). Recombinant IL-1 receptor type I,including human IL-1 receptor type I, for such assays is readilyavailable from a variety of commercial sources (see for example R&DSystems, SIGMA). IL-1 receptor type I also can be expressed from anexpression construct or vector introduced into an appropriate host cellusing standard molecular biology and transfection techniques known inthe art. The expressed IL-1 receptor type I may then be isolated andpurified for use in binding assays, or alternatively used directly in acell associated form.

For example, the binding of IL-1β to IL-1 receptor type I may bedetermined by immobilizing an IL-1β binding antibody, contacting IL-1βwith the immobilized antibody and determining whether the IL-1β wasbound to the antibody, and contacting a soluble form of IL-1RI with thebound IL-1β/antibody complex and determining whether the soluble IL-1RIwas bound to the complex. The protocol may also include contacting thesoluble IL-1RI with the immobilized antibody before the contact withIL-1β, to confirm that the soluble IL-1RI does not bind to theimmobilized antibody. This protocol can be performed using a Biacore®instrument for kinetic analysis of binding interactions. Such a protocolcan also be employed to determine whether an antibody or other moleculepermits or blocks the binding of IL-1β to IL-1 receptor type I.

For other IL-1β/IL-1RI binding assays, the permitting or blocking ofIL-1β binding to IL-1 receptor type I may be determined by comparing thebinding of IL-1β to IL-1RI in the presence or absence of IL-1βantibodies or IL-1β binding fragments thereof. Blocking is identified inthe assay readout as a designated reduction of IL-1β binding to IL-1receptor type I in the presence of anti-IL-1β antibodies or IL-1βbinding fragments thereof, as compared to a control sample that containsthe corresponding buffer or diluent but not an IL-1β antibody or IL-1βbinding fragment thereof. The assay readout may be qualitatively viewedas indicating the presence or absence of blocking, or may bequantitatively viewed as indicating a percent or fold reduction inbinding due to the presence of the antibody or fragment.

Alternatively or additionally, when an IL-1β binding antibody or IL-1βbinding fragment substantially blocks IL-1β binding to IL-1R1, the IL-1βbinding to IL-1RI is reduced by at least 10-fold, alternatively at leastabout 20-fold, alternatively at least about 50-fold, alternatively atleast about 100-fold, alternatively at least about 1000-fold,alternatively at least about 10000-fold, or more, compared to binding ofthe same concentrations of IL-1β and IL-1RI in the absence of theantibody or fragment. As another example, when an IL-1β binding antibodyor IL-1β binding fragment substantially permits IL-1β binding to IL-1R1,the IL-1β binding to IL-1RI is at least about 90%, alternatively atleast about 95%, alternatively at least about 99%, alternatively atleast about 99.9%, alternatively at least about 99.99%, alternatively atleast about 99.999%, alternatively at least about 99.9999%,alternatively substantially identical to binding of the sameconcentrations of IL-1β and IL-1RI in the absence of the antibody orfragment.

The present disclosure may in certain embodiments encompass IL-1βbinding antibodies or IL-1β binding fragments that bind to the sameepitope or substantially the same epitope as one or more of theexemplary antibodies described herein. Alternatively or additionally,the IL-1β binding antibodies or IL-1β binding fragments compete with thebinding of an antibody having variable region sequences of AB7,described in U.S. application Ser. No. 11/472,813 (sequences shownbelow). Alternatively or additionally, the present disclosureencompasses IL-1β binding antibodies and fragments that bind to anepitope contained in the amino acid sequence ESVDPKNYPKKKMEKRFVFNKIE(SEQ ID NO: 3). As contemplated herein, one can readily determine if anIL-1β binding antibody or fragment binds to the same epitope orsubstantially the same epitope as one or more of the exemplaryantibodies, such as for example the antibody designated AB7, using anyof several known methods in the art.

For example, the key amino acid residues (epitope) bound by an IL-1βbinding antibody or fragment may be determined using a peptide array,such as for example, a PepSpot™ peptide array (JPT Peptide Technologies,Berlin, Germany), wherein a scan of twelve amino-acid peptides, spanningthe entire IL-1β amino acid sequence, each peptide overlapping by 11amino acid to the previous one, is synthesized directly on a membrane.The membrane carrying the peptides is then probed with the antibody forwhich epitope binding information is sought, for example at aconcentration of 2 μg/ml, for 2 hr at room temperature. Binding ofantibody to membrane bound peptides may be detected using a secondaryHRP-conjugated goat anti-human (or mouse, when appropriate) antibody,followed by enhanced chemiluminescence (ECL). The peptides spot(s)corresponding to particular amino acid residues or sequences of themature IL-1β protein, and which score positive for antibody binding, areindicative of the epitope bound by the particular antibody.

Alternatively or in addition, antibody competition experiments may beperformed and such assays are well known in the art. For example, todetermine if an antibody or fragment binds to an epitope contained in apeptide sequence comprising the amino acids ESVDPKNYPKKKMEKRFVFNKIE (SEQID NO: 3), which corresponds to residues 83-105 of the mature IL-1βprotein, an antibody of unknown specificity may be compared with any ofthe exemplary of antibodies (e.g., AB7) of the present disclosure.Binding competition assays may be performed, for example, using aBiacore® instrument for kinetic analysis of binding interactions or byELISA. In such an assay, the antibody of unknown epitope specificity isevaluated for its ability to compete for binding against the knowncomparator antibody (e.g., AB7). Competition for binding to a particularepitope is determined by a reduction in binding to the IL-1β epitope ofat least about 50%, or at least about 70%, or at least about 80%, or atleast about 90%, or at least about 95%, or at least about 99% or about100% for the known comparator antibody (e.g., AB7) and is indicative ofbinding to substantially the same epitope.

In view of the identification in this disclosure of IL-1β bindingregions in exemplary antibodies and/or epitopes recognized by thedisclosed antibodies, it is contemplated that additional antibodies withsimilar binding characteristics and therapeutic or diagnostic utilitycan be generated that parallel the embodiments of this disclosure.

Antigen-binding fragments of an antibody include fragments that retainthe ability to specifically bind to an antigen, generally by retainingthe antigen-binding portion of the antibody. It is well established thatthe antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of antigen-bindingportions include (i) a Fab fragment, which is a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)² fragment,which is a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment which is theVH and CH1 domains; (iv) a Fv fragment which is the VL and VH domains ofa single arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which is a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso encompassed within the term antigen-binding portion of an antibody.The IL-1β binding antibodies and fragments of the present disclosurealso encompass monovalent or multivalent, or monomeric or multimeric(e.g. tetrameric), CDR-derived binding domains with or without ascaffold (for example, protein or carbohydrate scaffolding).

The present IL-1β binding antibodies or fragments may be part of alarger immunoadhesion molecules, formed by covalent or non-covalentassociation of the antibody or antibody portion with one or more otherproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas6:93-101) and use of a cysteine residue, a marker peptide and aC-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).Antibodies and fragments comprising immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.Preferred antigen binding portions are complete domains or pairs ofcomplete domains.

The IL-1β binding antibodies and fragments of the present disclosurealso encompass domain antibody (dAb) fragments (Ward et al., Nature341:544-546, 1989) which consist of a V_(H) domain. The IL-1β bindingantibodies and fragments of the present disclosure also encompassdiabodies, which are bivalent antibodies in which V_(H) and V_(L)domains are expressed on a single polypeptide chain, but using a linkerthat is too short to allow for pairing between the two domains on thesame chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., EP 404,097; WO 93/11161; Holliger et al., Proc. Natl. Acad. Sci.USA 90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123, 1994).Diabodies can be bispecific or monospecific.

The IL-1β binding antibodies and fragments of the present disclosurealso encompass single-chain antibody fragments (scFv) that bind toIL-1β. An scFv comprises an antibody heavy chain variable region (V_(H))operably linked to an antibody light chain variable region (V_(L))wherein the heavy chain variable region and the light chain variableregion, together or individually, form a binding site that binds IL-1β.An scFv may comprise a V_(H) region at the amino-terminal end and aV_(L) region at the carboxy-terminal end. Alternatively, scFv maycomprise a V_(L) region at the amino-terminal end and a V_(H) region atthe carboxy-terminal end. Furthermore, although the two domains of theFv fragment, V_(L) and V_(H), are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

An scFv may optionally further comprise a polypeptide linker between theheavy chain variable region and the light chain variable region. Suchpolypeptide linkers generally comprise between 1 and 50 amino acids,alternatively between 3 and 12 amino acids, alternatively 2 amino acids.An example of a linker peptide for linking heavy and light chains in anscFv comprises the 5 amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:6). Other examples comprise one or more tandem repeats of this sequence(for example, a polypeptide comprising two to four repeats ofGly-Gly-Gly-Gly-Ser (SEQ ID NO: 6) to create linkers.

The IL-1β binding antibodies and fragments of the present disclosurealso encompass heavy chain antibodies (HCAb). Exceptions to the H₂L₂structure of conventional antibodies occur in some isotypes of theimmunoglobulins found in camelids (camels, dromedaries and llamas;Hamers-Casterman et al., 1993 Nature 363: 446; Nguyen et al., 1998 J.Mol. Biol. 275: 413), wobbegong sharks (Nuttall et al., Mol. Immunol.38:313-26, 2001), nurse sharks (Greenberg et al., Nature 374:168-73,1995; Roux et al., 1998 Proc. Nat. Acad. Sci. USA 95: 11804), and in thespotted raffish (Nguyen, et al., “Heavy-chain antibodies in Camelidae; acase of evolutionary innovation,” 2002 Immunogenetics 54(1): 39-47).These antibodies can apparently form antigen-binding regions using onlyheavy chain variable regions, in that these functional antibodies aredimers of heavy chains only (referred to as “heavy-chain antibodies” or“HCAbs”). Accordingly, some embodiments of the present IL-1β bindingantibodies and fragments may be heavy chain antibodies that specificallybind to IL-1β. For example, heavy chain antibodies that are a class ofIgG and devoid of light chains are produced by animals of the genusCamelidae which includes camels, dromedaries and llamas(Hamers-Casterman et al., Nature 363:446-448 (1993)). HCAbs have amolecular weight of about 95 kDa instead of the about 160 kDa molecularweight of conventional IgG antibodies. Their binding domains consistonly of the heavy-chain variable domains, often referred to as V_(BH) todistinguish them from conventional V_(H). Muyldermans et al., J. Mol.Recognit. 12:131-140 (1999). The variable domain of the heavy-chainantibodies is sometimes referred to as a nanobody (Cortez-Retamozo etal., Cancer Research 64:2853-57, 2004). A nanobody library may begenerated from an immunized dromedary as described in Conrath et al.,(Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombinantmethods.

Since the first constant domain (C_(H1)) is absent (spliced out duringmRNA processing due to loss of a splice consensus signal), the variabledomain (V_(HH)) is immediately followed by the hinge region, the C_(H2)and the C_(H3) domains (Nguyen et al., Mol. Immunol. 36:515-524 (1999);Woolven et al., Immunogenetics 50:98-101 (1999)). Camelid V_(HH)reportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH₂, and CH3 domains and lack a CH1 domain (Hamers-Casterman etal., supra). For example, llama IgG1 is a conventional (H₂L₂) antibodyisotype in which V_(H) recombines with a constant region that containshinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 areheavy chain-only isotypes that lack CH1 domains and that contain nolight chains.

Although the HCAbs are devoid of light chains, they have anantigen-binding repertoire. The genetic generation mechanism of HCAbs isreviewed in Nguyen et al. Adv. Immunol 79:261-296 (2001) and Nguyen etal., Immunogenetics 54:39-47 (2002). Sharks, including the nurse shark,display similar antigen receptor-containing single monomeric V-domains.Irving et al., J. Immunol. Methods 248:31-45 (2001); Roux et al., Proc.Natl. Acad. Sci. USA 95:11804 (1998).

V_(HH) s comprise small intact antigen-binding fragments (for example,fragments that are about 15 kDa, 118-136 residues). Camelid V_(HH)domains have been found to bind to antigen with high affinity (Desmyteret al., J. Biol. Chem. 276:26285-90, 2001), with V_(HH) affinitiestypically in the nanomolar range and comparable with those of Fab andscFv fragments. V_(HH)s are highly soluble and more stable than thecorresponding derivatives of scFv and Fab fragments. V_(H) fragmentshave been relatively difficult to produce in soluble form, butimprovements in solubility and specific binding can be obtained whenframework residues are altered to be more V_(HH)-like. (See, forexample, Reichman et al., J Immunol Methods 1999, 231:25-38.) V_(HH)scarry amino acid substitutions that make them more hydrophilic andprevent prolonged interaction with BiP (immunoglobulin heavy-chainbinding protein), which normally binds to the H-chain in the EndoplasmicReticulum (ER) during folding and assembly, until it is displaced by theL-chain. Because of the V_(HH)s′ increased hydrophilicity, secretionfrom the ER is improved.

Functional V_(HH)s may be obtained by proteolytic cleavage of HCAb of animmunized camelid, by direct cloning of V_(HH) genes from B-cells of animmunized camelid resulting in recombinant V_(HH)s, or from naive orsynthetic libraries. V_(HH)s with desired antigen specificity may alsobe obtained through phage display methodology. Using V_(HH)s in phagedisplay is much simpler and more efficient compared to Fabs or scFvs,since only one domain needs to be cloned and expressed to obtain afunctional antigen-binding fragment. Muyldermans, Biotechnol. 74:277-302(2001); Ghahroudi et al., FEBS Lett. 414:521-526 (1997); and van derLinden et al., J. Biotechnol. 80:261-270 (2000). Methods for generatingantibodies having camelid heavy chains are also described in U.S. PatentPublication Nos. 20050136049 and 20050037421.

Ribosome display methods may be used to identify and isolate scFv and/orV_(HH) molecules having the desired binding activity and affinity.Irving et al., J. Immunol. Methods 248:31-45 (2001). Ribosome displayand selection has the potential to generate and display large libraries(10¹⁴).

Other embodiments provide V_(HH)-like molecules generated through theprocess of camelisation, by modifying non-Camelidae V_(H)s, such ashuman V_(HH)s, to improve their solubility and prevent non-specificbinding. This is achieved by replacing residues on the V_(L)s side ofV_(H)s with V_(HH)-like residues, thereby mimicking the more solubleV_(HH) fragments. Camelised V_(H) fragments, particularly those based onthe human framework, are expected to exhibit a greatly reduced immuneresponse when administered in vivo to a patient and, accordingly, areexpected to have significant advantages for therapeutic applications.Davies et al., FEBS Lett. 339:285-290 (1994); Davies et al., ProteinEng. 9:531-537 (1996); Tanha et al., J. Biol. Chem. 276:24774-24780(2001); and Riechmann et al., Immunol. Methods 231:25-38 (1999).

A wide variety of expression systems are available for the production ofIL-1β fragments including Fab fragments, scFv, and V_(HH)s. For example,expression systems of both prokaryotic and eukaryotic origin may be usedfor the large-scale production of antibody fragments and antibody fusionproteins. Particularly advantageous are expression systems that permitthe secretion of large amounts of antibody fragments into the culturemedium.

Production of bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH₁ (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab. A “minibody”consisting of scFv fused to CH3 via a peptide linker (hingeless) or viaan IgG hinge has been described in Olafsen, et al., Protein Eng Des Sel.2004 April; 17(4):315-23.

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA.101:17616-21, 2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody construct in intracellular regions, may beproduced as described in Mhashilkar et al., (EMBO J. 14:1542-51, 1995)and Wheeler et al. (FASEB J. 17:1733-5, 2003). Transbodies arecell-permeable antibodies in which a protein transduction domains (PTD)is fused with single chain variable fragment (scFv) antibodies Heng etal., (Med Hypotheses. 64:1105-8, 2005).

The IL-1β binding antibodies and fragments of the present disclosurealso encompass antibodies that are SMIPs or binding domainimmunoglobulin fusion proteins specific for target protein. Theseconstructs are single-chain polypeptides comprising antigen bindingdomains fused to immunoglobulin domains necessary to carry out antibodyeffector functions. See e.g., WO03/041600, U.S. Patent publication20030133939 and US Patent Publication 20030118592.

The IL-1β binding antibodies and fragments of the present disclosurealso encompass immunoadhesins. One or more CDRs may be incorporated intoa molecule either covalently or noncovalently to make it animmunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of alarger polypeptide chain, may covalently link the CDR(s) to anotherpolypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRsdisclosed herein permit the immunoadhesin to specifically bind toIL-1f3.

The IL-1β binding antibodies and fragments of the present disclosurealso encompass antibody mimics comprising one or more IL-1β bindingportions built on an organic or molecular scaffold (such as a protein orcarbohydrate scaffold). Proteins having relatively definedthree-dimensional structures, commonly referred to as protein scaffolds,may be used as reagents for the design of antibody mimics. Thesescaffolds typically contain one or more regions which are amenable tospecific or random sequence variation, and such sequence randomizationis often carried out to produce libraries of proteins from which desiredproducts may be selected. For example, an antibody mimic can comprise achimeric non-immunoglobulin binding polypeptide having animmunoglobulin-like domain containing scaffold having two or moresolvent exposed loops containing a different CDR from a parent antibodyinserted into each of the loops and exhibiting selective bindingactivity toward a ligand bound by the parent antibody.Non-immunoglobulin protein scaffolds have been proposed for obtainingproteins with novel binding properties. (Tramontano et al., J. Mol.Recognit. 7:9, 1994; McConnell and Hoess, J. Mol. Biol. 250:460, 1995).Other proteins have been tested as frameworks and have been used todisplay randomized residues on alpha helical surfaces (Nord et al., Nat.Biotechnol. 15:772, 1997; Nord et al., Protein Eng. 8:601, 1995), loopsbetween alpha helices in alpha helix bundles (Ku and Schultz, Proc.Natl. Acad. Sci. USA 92:6552, 1995), and loops constrained by disulfidebridges, such as those of the small protease inhibitors (Markland etal., Biochemistry 35:8045, 1996; Markland et al., Biochemistry 35:8058,1996; Rottgen and Collins, Gene 164:243, 1995; Wang et al., J. Biol.Chem. 270:12250, 1995). Methods for employing scaffolds for antibodymimics are disclosed in U.S. Pat. No. 5,770,380 and US PatentPublications 2004/0171116, 2004/0266993, and 2005/0038229.

The anti-IL-1β binding antibodies or binding fragments thereof for usein the methods herein generally bind to IL-1β with high affinity (e.g.,as determined with BIACORE). In preferred embodiments, the antibody orfragment thereof binds to IL-1β with an equilibrium binding dissociationconstant (K_(D)) of about 10 nM or less, about 5 nM or less, about 1 nMor less, about 500 pM or less, about 250 pM or less, about 100 pM orless, about 50 pM or less, or about 25 pM or less. In particularlypreferred embodiments, the antibody or antibody fragment binds to humanIL-1β with a dissociation constant of about 100 pM or less, about 50 pMor less, about 10 pM or less, about 5 pM or less, about 3 pM or less,about 1 pM or less, about 0.75 pM or less, about 0.5 pM or less, about0.3 pM or less, about 0.2 pM or less, or about 0.1 pM or less. Inparticularly preferred embodiments, the antibody or antibody fragmentbinds to human IL-1β with a dissociation constant of about 10 pM orless.

Antibodies or fragments of the present disclosure may, for example, bindto IL-1β with an IC₅₀ of about 10 nM or less, about 5 nM or less, about2 nM or less, about 1 nM or less, about 0.75 nM or less, about 0.5 nM orless, about 0.4 nM or less, about 0.3 nM or less, or even about 0.2 nMor less, as determined by enzyme linked immunosorbent assay (ELISA).Preferably, the antibody or antibody fragment of the present disclosuredoes not cross-react with any target other than IL-1. For example, thepresent antibodies and fragments may bind to IL-1β, but do notdetectably bind to IL-1α, or have at least about 100 times (e.g., atleast about 150 times, at least about 200 times, or even at least about250 times) greater selectivity in its binding of IL-1β relative to itsbinding of IL-1α. Antibodies or fragments used according to the presentdisclosure may, in certain embodiments, inhibit IL-1β induced expressionof serum IL-6 in an animal by at least 50% (e.g., at least 60%, at least70%, or even at least 80%) as compared to the level of serum IL-6 in anIL-1β stimulated animal that has not been administered an antibody orfragment of the present disclosure. Antibodies may bind IL-1β but permitor substantially permit the binding of the bound IL-1β ligand to IL-1receptor type I (IL-1R1). In contrast to many known IL-1β bindingantibodies that block or substantially interfere with binding of IL-1βto IL-1R1, the antibodies designated AB5 and AB7 (U.S. application Ser.No. 11/472,813) selectively bind to the IL-1β ligand, but permit thebinding of the bound IL-1β ligand to IL-1R1. For example, the antibodydesignated AB7 binds to an IL-1β epitope but still permits the boundIL-1β to bind to IL-1R1. In certain embodiments, the antibody maydecrease the affinity of interaction of bound IL-1β to bind to IL-1R1.Accordingly, the disclosure provides, in a related aspect, use of anIL-1β binding antibody or IL-1β binding antibody fragment that has atleast one of the aforementioned characteristics. Any of the foregoingantibodies, antibody fragments, or polypeptides of the disclosure can behumanized or human engineered, as described herein.

A variety of IL-1 (e.g., IL-1β) antibodies and fragments known in theart may be used according the methods provided herein, including forexample antibodies described in or derived using methods described inthe following patents and patent applications: U.S. Pat. No. 4,935,343;US 2003/0026806; US 2003/0124617 (e.g., antibody AAL160); WO 2006/081139(e.g., antibody 9.5.2); WO 03/034984; WO 95/01997 (e.g., antibodySK48-E26 VTKY); U.S. Pat. No. 7,446,175 (e.g., antibody ACZ 885); WO03/010282 (e.g., antibody Hu007); WO 03/073982 (e.g., antibody N55S),U.S. Pat. No. 7,541,033 (e.g., W17, U43, W13, W18, W20), U.S. Pat. No.7,491,392, WO 2004/072116, WO 2004/067568, EP 0 267 611 B1, EP 0 364 778B1, and U.S. application Ser. No. 11/472,813. As a non-limiting example,antibodies AB5 and AB7 (U.S. application Ser. No. 11/472,813,WO2007/002261) may be used in accordance with the present disclosure.Variable region sequences of AB5 and AB7 are as follows:

AB7 LIGHT CHAIN (SEQ ID NO: 1)DIQMTQSTSSLSASVGDRVTITCRASQDISNYLSWYQQKPGKAVKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCLQGKMLPW TFGQGTKLEIKThe underlined sequences depict (from left to right) CDR1, 2 and 3.

HEAVY CHAIN (SEQ ID NO: 2)QVQLQESGPGLVKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLE WLAHIWWDGDESYNPSLKSRLTISKDTSKNQVSLKITSVTAADTAVYF CARNRYDPPWFVDWGQGTLVTVSSThe underlined sequences depict (from left to right) CDR1, 2 and 3.

AB5 LIGHT CHAIN (SEQ ID NO: 4)DIQMTQTTSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYYTSKLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCLQGKMLPW TFGGGTKLEIKThe underlined sequences depict (from left to right) CDR1, 2 and 3.

HEAVY CHAIN (SEQ ID NO: 5)QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDGDESYNPSLKTQLTISKDTSRNQVFLKITSVDTVDTATYFCARNRYDPPWFVDWGQGTLVTVSSThe underlined sequences depict (from left to right) CDR1, 2 and 3.

In some embodiments, IL-1β antibodies or fragments thereof for use inany and/or all of the methods disclosed herein may bind to human IL-1βwith a dissociation constant of about 1 nM or less. In some embodiments,the antibody or fragment binds to human IL-1β with a dissociationconstant of about 500 pM or less. In some embodiments, the anti-IL-1βbinding antibody or binding fragment thereof binds to human IL-1β with adissociation constant of about 250 pM or less. In some embodiments, theanti-IL-1β binding antibody or binding fragment thereof binds to humanIL-1β with a dissociation constant of about 100 pM or less. In someembodiments, the antibody or fragment binds to human IL-1β with adissociation constant of about 50 pM or less. In some embodiments, theantibody or fragment binds to human IL-1β with a dissociation constantof about 10 pM or less. In some embodiments, the antibody or fragmentbinds to human IL-1β with a dissociation constant of about 5 pM or less.In some embodiments, the antibody or fragment binds to human IL-1β witha dissociation constant of about 1 pM or less. In some embodiments, theantibody or fragment binds to human IL-1β with a dissociation constantof about 0.3 pM or less.

In some embodiments, the anti-IL-1β binding antibody or binding fragmentthereof is a neutralizing antibody.

In some embodiments, the anti-IL-1β binding antibody or binding fragmentthereof binds to an IL-1β epitope such that the bound antibody orfragment substantially permits the binding of IL-1β to IL-1 receptor I(IL-1R1).

In some embodiments, the anti-IL-1β binding antibody or binding fragmentthereof does not detectably bind to IL-1α, IL-1R or IL-1Ra.

In some embodiments, the anti-IL-1β binding antibody or binding fragmentthereof competes with the binding of an antibody having the light chainvariable region of SEQ ID NO:1 and the heavy chain variable region ofSEQ ID NO:2. In some embodiments of any of the methods described above,the anti-IL-1β binding antibody or binding fragment thereof binds to anepitope that is the same or substantially the same as an epitope that isbound by an antibody having the light chain variable region of SEQ IDNO:1 and the heavy chain variable region of SEQ ID NO:2. In someembodiments of any of the methods described above, the anti-IL-1βbinding antibody or binding fragment thereof comprises a light chainvariable region of SEQ ID NO:1 and a heavy chain variable region of SEQID NO:2.

In some embodiments, the anti-IL-1β binding antibody or binding fragmentthereof binds to an epitope incorporating Glu64 of IL-1β.

In some embodiments, the anti-IL-1β binding antibody or binding fragmentthereof binds to amino acids 1-34 of the N terminus of IL-1β.

In some embodiments, the anti-IL-1β binding antibody or binding fragmentthereof is humanized or human.

The present disclosure also provides uses of an anti-IL-1β bindingantibody or binding fragment thereof which has a lower IC₅₀ than anIL-1β receptor antagonist in a human whole blood IL-1β inhibition assaythat measures IL-1β induced production of IL-8, in the manufacture of acomposition for use in the reduction, prevention or treatment of acardiac event or a cardiovascular disease.

In another aspect, the methods comprise administering a therapeuticallyeffective amount of an anti-IL-1β antibody or fragment thereof, whereinthe antibody or fragment thereof has a lower IC₅₀ than an IL-1β receptorantagonist in a human whole blood IL-1β inhibition assay that measuresIL-1β induced production of IL-8. In one embodiment, the antibody orfragment has an IC₅₀ that is less than about 90%, 80%, 70%, 60%, 50% ofthe IC₅₀ of an IL-1β receptor antagonist in a human whole blood IL-1βinhibition assay that measures IL-1β induced production of IL-8. In afurther embodiment, the antibody or fragment has an IC₅₀ that is lessthan about 40%, 30%, 20%, 10% of the IC₅₀ of an IL-1β receptorantagonist in a human whole blood IL-1β inhibition assay that measuresIL-1β induced production of IL-8. In a preferred embodiment, theantibody or fragment has an IC₅₀ that is less than about 8%, 5%, 4%, 3%,2%, 1% of the IC₅₀ of an IL-1β receptor antagonist in a human wholeblood IL-1β inhibition assay that measures IL-1β induced production ofIL-8. In one embodiment, the IL-1β receptor antagonist is anakinra(i.e., Kineret®).

In another aspect, the method provided herein comprises administering atherapeutically effective amount of an anti-IL-1β antibody or fragmentthereof to the subject, wherein the antibody or fragment thereofprovides in vivo inhibition of IL-1β stimulated release of IL-6 in micecompared to a control antibody using an assay that is described byEconomides et al., Nature Med., 9:47-52 (2003) which is incorporated byreference. In one embodiment the antibody or fragment provides in vivoinhibition of IL-1β stimulated release of IL-6 in mice of at least about10%, 20%, 30%, 40%, 50% compared to the control antibody. In a furtherembodiment, the antibody or fragment provides in vivo inhibition ofIL-1β stimulated release of IL-6 in mice of at least about 60%, 70%,80%, 90%, 95% compared to the control antibody. In one embodiment, thecontrol antibody is an isotype control antibody.

In another aspect, the disclosure provides a method comprisingadministering a therapeutically effective amount of an anti-IL-1βantibody or fragment thereof to the human, wherein the antibody orfragment thereof inhibits Staphylococcus epidermidis induced cytokineproduction in human whole blood compared to a control where no antibodyis used. In one embodiment the antibody or fragment provides a greaterlevel of inhibition of Staphylococcus epidermidis induced cytokineproduction in human whole blood by at least about 10%, 20%, 30%, 40%,50% compared to the control. In a further embodiment, the antibody orfragment provides a greater level of inhibition of Staphylococcusepidermidis induced cytokine production in human whole blood by at leastabout 60%, 70%, 80%, 90%, 95% compared to the control. In oneembodiment, the inhibited cytokines are IL-1β, IL-1a, IL-6, IL-8,IL-1Ra, TNFα or IFNγ.

The antibodies and antibody fragments described herein can be preparedby any suitable method. Suitable methods for preparing such antibodiesand antibody fragments are known in the art. Other methods for preparingthe antibodies and antibody fragments are as described herein as part ofthe disclosure. The antibody, antibody fragment, or polypeptide of thepresent disclosure, as described herein, can be isolated or purified toany degree. As used herein, an isolated compound is a compound that hasbeen removed from its natural environment. A purified compound is acompound that has been increased in purity, such that the compoundexists in a form that is more pure than it exists (i) in its naturalenvironment or (ii) when initially synthesized and/or amplified underlaboratory conditions, wherein “purity” is a relative term and does notnecessarily mean “absolute purity.”

Pharmaceutical Compositions

IL-1 (e.g., IL-1β) binding antibodies and antibody fragments for useaccording to the present disclosure can be formulated in compositions,especially pharmaceutical compositions, for use in the methods herein.Such compositions comprise a therapeutically or prophylacticallyeffective amount of an IL-1β binding antibody or antibody fragment ofthe disclosure in admixture with a suitable carrier, e.g., apharmaceutically acceptable agent. Typically, IL-1β binding antibodiesand antibody fragments of the disclosure are sufficiently purified foradministration to an animal before formulation in a pharmaceuticalcomposition.

Pharmaceutically acceptable agents include carriers, excipients,diluents, antioxidants, preservatives, coloring, flavoring and dilutingagents, emulsifying agents, suspending agents, solvents, fillers,bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents,wetting agents, complexing agents, buffering agents, antimicrobials, andsurfactants.

Neutral buffered saline or saline mixed with albumin are exemplaryappropriate carriers. The pharmaceutical compositions can includeantioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, pluronics, or polyethylene glycol (PEG). Alsoby way of example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol, and the like. Suitable preservatives include benzalkoniumchloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid and the like. Hydrogen peroxide also can beused as preservative. Suitable cosolvents include glycerin, propyleneglycol, and PEG. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal, and the like. Thebuffers can be conventional buffers such as acetate, borate, citrate,phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH4-5.5, and Tris buffer can be about pH 7-8.5. Additional pharmaceuticalagents are set forth in Remington's Pharmaceutical Sciences, 18thEdition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

The composition can be in liquid form or in a lyophilized orfreeze-dried form and may include one or more lyoprotectants,excipients, surfactants, high molecular weight structural additivesand/or bulking agents (see for example U.S. Pat. Nos. 6,685,940,6,566,329, and 6,372,716). In one embodiment, a lyoprotectant isincluded, which is a non-reducing sugar such as sucrose, lactose ortrehalose. The amount of lyoprotectant generally included is such that,upon reconstitution, the resulting formulation will be isotonic,although hypertonic or slightly hypotonic formulations also may besuitable. In addition, the amount of lyoprotectant should be sufficientto prevent an unacceptable amount of degradation and/or aggregation ofthe protein upon lyophilization. Exemplary lyoprotectant concentrationsfor sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilizedformulation are from about 10 mM to about 400 mM. In another embodiment,a surfactant is included, such as for example, nonionic surfactants andionic surfactants such as polysorbates (e.g. polysorbate 20, polysorbate80); poloxamers (e.g. poloxamer 188); poly (ethylene glycol) phenylethers (e.g. Triton); sodium dodecyl sulfate (SDS); sodium laurelsulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc). Exemplaryamounts of surfactant that may be present in the pre-lyophilizedformulation are from about 0.001-0.5%. High molecular weight structuraladditives (e.g. fillers, binders) may include for example, acacia,albumin, alginic acid, calcium phosphate (dibasic), cellulose,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,dextrin, dextrates, sucrose, tylose, pregelatinized starch, calciumsulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose,disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite,polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose,compressible sugar, magnesium aluminum silicate, maltodextrin,polyethylene oxide, polymethacrylates, povidone, sodium alginate,tragacanth microcrystalline cellulose, starch, and zein. Exemplaryconcentrations of high molecular weight structural additives are from0.1% to 10% by weight. In other embodiments, a bulking agent (e.g.,mannitol, glycine) may be included.

Compositions can be suitable for parenteral administration. Exemplarycompositions are suitable for injection or infusion into an animal byany route available to the skilled worker, such as intraarticular,subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral(intraparenchymal), intracerebroventricular, intramuscular, intraocular,intraarterial, intralesional, intrarectal, transdermal, oral, andinhaled routes. A parenteral formulation typically will be a sterile,pyrogen-free, isotonic aqueous solution, optionally containingpharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringers'dextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, anti-microbials, anti-oxidants, chelating agents, inertgases and the like. See generally, Remington's Pharmaceutical Science,16th Ed., Mack Eds., 1980, which is incorporated herein by reference.

Pharmaceutical compositions described herein can be formulated forcontrolled or sustained delivery in a manner that provides localconcentration of the product (e.g., bolus, depot effect) sustainedrelease and/or increased stability or half-life in a particular localenvironment. The present disclosure contemplates that in certainembodiments such compositions may include a significantly larger amountof antibody or fragment in the initial deposit, while the effectiveamount of antibody or fragment actually released and available at anypoint in time for is in accordance with the disclosure herein an amountmuch lower than the initial deposit. The compositions can include theformulation of IL-1β binding antibodies, antibody fragments, nucleicacids, or vectors of the disclosure with particulate preparations ofpolymeric compounds such as polylactic acid, polyglycolic acid, etc., aswell as agents such as a biodegradable matrix, injectable microspheres,microcapsular particles, microcapsules, bioerodible particles beads,liposomes, and implantable delivery devices that provide for thecontrolled or sustained release of the active agent which then can bedelivered as a depot injection. Techniques for formulating suchsustained- or controlled-delivery means are known and a variety ofpolymers have been developed and used for the controlled release anddelivery of drugs. Such polymers are typically biodegradable andbiocompatible. Polymer hydrogels, including those formed by complexationof enantiomeric polymer or polypeptide segments, and hydrogels withtemperature or pH sensitive properties, may be desirable for providingdrug depot effect because of the mild and aqueous conditions involved intrapping bioactive protein agents (e.g., antibodies). See, for example,the description of controlled release porous polymeric microparticlesfor the delivery of pharmaceutical compositions in PCT ApplicationPublication WO 93/15722.

Suitable materials for this purpose include polylactides (see, e.g.,U.S. Pat. No. 3,773,919), polymers of poly-(α-hydroxycarboxylic acids),such as poly-D-(−)-3-hydroxybutyric acid (EP 133,988A), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al.,J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyricacid. Other biodegradable polymers include poly(lactones),poly(acetals), poly(orthoesters), and poly(orthocarbonates).Sustained-release compositions also may include liposomes, which can beprepared by any of several methods known in the art (see, e.g., Eppsteinet al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrieritself, or its degradation products, should be nontoxic in the targettissue and should not further aggravate the condition. This can bedetermined by routine screening in animal models of the target disorderor, if such models are unavailable, in normal animals.

Microencapsulation of recombinant proteins for sustained release hasbeen performed successfully with human growth hormone (rhGH),interferon-(rhIFN—), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology. 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010. The sustained-release formulations of these proteins weredeveloped using poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be depending on its molecular weight and composition. Lewis,“Controlled release of bioactive agents from lactide/glycolide polymer,”in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as DrugDelivery Systems (Marcel Dekker: New York, 1990), pp. 1-41. Additionalexamples of sustained release compositions include, for example, EP58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No.1176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al.,Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. Release 90, 261[2003], Zhu et al., Nat. Biotechnol. 18, 24 [2000], and Dai et al.,Colloids Surf B Biointerfaces 41, 117 [2005].

Bioadhesive polymers are also contemplated for use in or withcompositions of the present disclosure. Bioadhesives are synthetic andnaturally occurring materials able to adhere to biological substratesfor extended time periods. For example, Carbopol and polycarbophil areboth synthetic cross-linked derivatives of poly(acrylic acid).Bioadhesive delivery systems based on naturally occurring substancesinclude for example hyaluronic acid, also known as hyaluronan.Hyaluronic acid is a naturally occurring mucopolysaccharide consistingof residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acidis found in the extracellular tissue matrix of vertebrates, including inconnective tissues, as well as in synovial fluid and in the vitreous andaqueous humour of the eye. Esterified derivatives of hyaluronic acidhave been used to produce microspheres for use in delivery that arebiocompatible and biodegrable (see for example, Cortivo et al.,Biomaterials (1991) 12:727-730; European Publication No. 517,565;International Publication No. WO 96/29998; Illum et al., J. ControlledRel. (1994) 29:133-141). Exemplary hyaluronic acid containingcompositions of the present disclosure comprise a hyaluronic acid esterpolymer in an amount of approximately 0.1% to about 40% (w/w) of anIL-1β binding antibody or fragment to hyaluronic acid polymer.

Both biodegradable and non-biodegradable polymeric matrices can be usedto deliver compositions in accordance with the present disclosure, andsuch polymeric matrices may comprise natural or synthetic polymers.Biodegradable matrices are preferred. The period of time over whichrelease occurs is based on selection of the polymer. Typically, releaseover a period ranging from between a few hours and three to twelvemonths is most desirable. Exemplary synthetic polymers which can be usedto form the biodegradable delivery system include: polymers of lacticacid and glycolic acid, polyamides, polycarbonates, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates,polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinylhalides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyanhydrides, polyurethanes and co-polymers thereof, poly(butic acid),poly(valeric acid), alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene and polyvinylpyrrolidone. Exemplary naturalpolymers include alginate and other polysaccharides including dextranand cellulose, collagen, chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), albumin and other hydrophilic proteins, zeinand other prolamines and hydrophobic proteins, copolymers and mixturesthereof. In general, these materials degrade either by enzymatichydrolysis or exposure to water in vivo, by surface or bulk erosion. Thepolymer optionally is in the form of a hydrogel (see for example WO04/009664, WO 05/087201, Sawhney, et al., Macromolecules, 1993, 26,581-587,) that can absorb up to about 90% of its weight in water andfurther, optionally is cross-linked with multi-valent ions or otherpolymers.

Delivery systems also include non-polymer systems that are lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono- di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the product is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in whicha product permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.Liposomes containing the product may be prepared by methods knownmethods, such as for example (DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324).

A pharmaceutical composition comprising an IL-1β binding antibody orfragment can be formulated for inhalation, such as for example, as a drypowder. Inhalation solutions also can be formulated in a liquefiedpropellant for aerosol delivery. In yet another formulation, solutionsmay be nebulized. Additional pharmaceutical composition for pulmonaryadministration include, those described, for example, in PCT ApplicationPublication WO 94/20069, which discloses pulmonary delivery ofchemically modified proteins. For pulmonary delivery, the particle sizeshould be suitable for delivery to the distal lung. For example, theparticle size can be from 1 μm to 5 μm; however, larger particles may beused, for example, if each particle is fairly porous.

Certain formulations containing IL-1β binding antibodies or antibodyfragments can be administered orally. Formulations administered in thisfashion can be formulated with or without those carriers customarilyused in the compounding of solid dosage forms such as tablets andcapsules. For example, a capsule can be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. Additional agents can be included to facilitate absorption ofa selective binding agent. Diluents, flavorings, low melting pointwaxes, vegetable oils, lubricants, suspending agents, tabletdisintegrating agents, and binders also can be employed.

Another preparation can involve an effective quantity of an IL-1βbinding antibody or fragment in a mixture with non-toxic excipientswhich are suitable for the manufacture of tablets. By dissolving thetablets in sterile water, or another appropriate vehicle, solutions canbe prepared in unit dose form. Suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Suitable and/or preferred pharmaceutical formulations can be determinedin view of the present disclosure and general knowledge of formulationtechnology, depending upon the intended route of administration,delivery format, and desired dosage. Regardless of the manner ofadministration, an effective dose can be calculated according to patientbody weight, body surface area, or organ size. Further refinement of thecalculations for determining the appropriate dosage for treatmentinvolving each of the formulations described herein are routinely madein the art and is within the ambit of tasks routinely performed in theart. Appropriate dosages can be ascertained through use of appropriatedose-response data.

Additional formulations will be evident in light of the presentdisclosure, including formulations involving IL-1β binding antibodiesand fragments in combination with one or more other therapeutic agents.For example, in some formulations, an IL-1β binding antibody, antibodyfragment, nucleic acid, or vector of the disclosure is formulated with asecond inhibitor of an IL-1 signaling pathway Representative secondinhibitors include, but are not limited to, antibodies, antibodyfragments, peptides, polypeptides, compounds, nucleic acids, vectors andpharmaceutical compositions, such as, for example, those described inU.S. Pat. No. 6,899,878, US 2003022869, US 20060094663, US 20050186615,US 20030166069, WO/04022718, WO/05084696, WO/05019259. For example, acomposition may comprise an IL-1β binding antibody, antibody fragment,nucleic acid, or vector of the disclosure in combination with an IL-1βbinding antibody, fragment, or a nucleic acid or vector encoding such anantibody or fragment.

Methods of Use

Anti-IL-1β binding antibodies or binding fragments thereof in atherapeutically effective amount may be used as disclosed by the methodsherein for the treatment and/or prevention of cardiovascular disease,including, for example, acute cardiovascular disease or chroniccardiovascular disease. Such methods, as well as pharmaceuticalcompositions for use in such methods, may be used for reducing, treatingor preventing a cardiovascular event, such as myocardial infarction,stroke, cardiovascular death, congestive heart failure, cardiac arrest,acute coronary syndrome, angina, or a revascularization procedure in asubject, including in a subject with a history of a risk factor forcardiovascular disease. The methods and pharmaceutical compositions mayalso be used to reduce mortality following a cardiovascular event in asubject. The present disclosure also contemplates the use of other IL-1pathway inhibitors, as an alternative or in addition to the anti-IL-1βantibodies or fragments.

The terms “prevention”, “prevent”, “preventing”, “suppression”,“suppress”, “suppressing”, “inhibit” and “inhibition” as used hereinwith respect to methods as described refer to preventing, suppressing orreducing, either temporarily or permanently, the onset of a clinicalsymptoms or manifestation of an event, disease or condition, such as,for example, a cardiovascular event or disease, (e.g., acute or chroniccardiovascular disease). Such preventing, suppressing or reducing neednot be absolute to be useful.

The terms “reduce”, “reducing” and “reduction” as used herein withrespect to the methods as described refer to delaying the time to anevent or disease, decreasing the likelihood or risk of an event ordisease, decreasing the incidence of an event or disease (e.g., in atreatment group), preventing the occurrence of an event or disease(e.g., prevention of a cardiovascular event or disease), decreasing themagnitude or severity of an event or disease (except in the case wherethe event is death), and/or decreasing the time to recovery from anevent or disease (except in the case where the event is death), such as,for example, treating or treatment of a cardiovascular event or disease,(e.g., acute or chronic cardiovascular disease).

The phrase “mortality following a cardiovascular event” as used hereinrefers to mortality (i.e., death) that occurs after a cardiovascularevent (e.g., after initiation of the cardiovascular event), and whichmay be, but need not be, directly or indirectly caused by or influencedby the cardiovascular event. Following also refers to the proximity intime (e.g., measurable) between the cardiovascular event and mortality,regardless of whether or not a direct or indirect causal link can bedetermined. The proximity in time between the cardiovascular event andmortality may vary and include an amount of time that is approachingbeing simultaneous.

The terms “treatment”, “treat” and “treating” as used with respect tomethods as described herein refers eliminating, reducing, suppressing orameliorating, either temporarily or permanently, a clinical symptom,manifestation or progression of an event, disease or condition, such as,for example, a cardiovascular event or disease, (e.g., acute or chroniccardiovascular disease). Such treating need not be absolute to beuseful.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver that a patient requires or will benefit from treatment.This judgment is made based on a variety of factors that are in therealm of a caregiver's expertise, but that includes the knowledge thatthe patient is ill, or will be ill, as the result of a condition that istreatable by a method or compound of the disclosure.

The term “in need of prevention” as used herein refers to a judgmentmade by a caregiver that a patient requires or will benefit fromprevention. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that includes the knowledgethat the patient will be ill or may become ill, as the result of acondition that is preventable by a method or compound of the disclosure.

The term “therapeutically effective amount” as used herein refers to anamount of a compound (e.g., antibody), either alone or as a part of apharmaceutical composition, that is capable of having any detectable,positive effect on any symptom, aspect, or characteristics of a diseasestate or condition when administered to a subject (e.g., as one or moredoses), including, for example, reducing a cardiovascular event ordisease, or reducing mortality following a cardiovascular event ordisease, (e.g., acute or chronic cardiovascular disease). Such effectneed not be absolute to be beneficial.

The present disclosure provides methods of treating a subject withcardiovascular disease, including, for example, acute cardiovasculardisease or chronic cardiovascular disease, comprising administering tosaid subject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof.

The present disclosure provides methods of reducing a cardiovascularevent in a subject, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof, wherein the subject is a subject with ahistory of a previous cardiovascular event or a history of at least onerisk factor for cardiovascular disease, and wherein the cardiovascularevent is myocardial infarction, stroke, cardiovascular death, congestiveheart failure, cardiac arrest, acute coronary syndrome, angina, or arevascularization procedure.

The present disclosure also provides methods of reducing acardiovascular event (e.g., delaying time to event, reducing likelihoodor risk of event, preventing an event, reducing severity of event,reducing time to recovery) in a subject with a history of at least onerisk factor for cardiovascular disease, comprising administering to saidsubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof, and wherein the cardiovascularevent is myocardial infarction, stroke, cardiovascular death, congestiveheart failure, cardiac arrest, acute coronary syndrome, angina, or arevascularization procedure.

The present disclosure also provides methods of reducing acardiovascular event (e.g., delaying time to event, reducing likelihoodor risk of event, preventing an event, reducing severity of event,reducing time to recovery) in a subject with a history of a previouscardiovascular event, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof, and wherein said cardiovascular event ismyocardial infarction, stroke, cardiovascular death, congestive heartfailure, cardiac arrest, acute coronary syndrome, angina or arevascularization procedure. In some embodiments, the previouscardiovascular event is a first cardiovascular event. In someembodiments, the previous or first cardiovascular event is selected fromthe group consisting of myocardial infarction, stroke, congestive heartfailure, acute coronary syndrome, angina and a revascularizationprocedure.

In some embodiments, the previous or first cardiovascular event ismyocardial infarction or acute coronary syndrome. In some embodiments,the myocardial infarction is myocardial infarction with ST elevation(e.g., ST-segment elevation myocardial infarction, STEMI). In someembodiments, the myocardial infarction is myocardial infarction withoutST elevation (e.g., non-ST-segment elevation myocardial infarction,NSTEMI). In some embodiments the presence or absence of ST elevation isdetermined by electrocardiogram (e.g., ECG, EKG).

In some embodiments, the method of reducing a cardiovascular event is amethod of reducing a second or subsequent cardiovascular event. In someembodiments, the cardiovascular event (e.g., second or subsequentcardiovascular event) is selected from the group consisting ofmyocardial infarction, stroke, cardiovascular death, congestive heartfailure, cardiac arrest, acute coronary syndrome, angina and arevascularization procedure. In some embodiments, the firstcardiovascular event and second cardiovascular event are the same typesof cardiovascular events. In some embodiments, the first cardiovascularevent and second cardiovascular event are different types ofcardiovascular events.

In some embodiments, the revascularization procedure is a coronary,carotid or peripheral arterial revascularization procedure. In someembodiments, the coronary, carotid or peripheral arterialrevascularization procedure is a percutaneous coronary intervention(PCI), a stent implant, coronary artery bypass graft (CABG), carotidendarterectomy, peripheral vascular disease bypass surgery, orperipheral angioplasty surgery.

In some preferred embodiments, the therapeutically effective amount ofan anti-IL-1β binding antibody or binding fragment thereof is firstadministered within 1 week of the cardiovascular event, within 96 hoursof the cardiovascular event, within 72 hours of the cardiovascularevent, within 48 hours of the cardiovascular event, within 24 hours ofthe cardiovascular event, or within 12 hours of the cardiovascularevent. In some embodiments, the subject also has a history of at leastone risk factor for cardiovascular disease. In some embodiments, therisk factor is manifest coronary heart disease, coronary artery disease,thrombosis, transient ischaemic attack, left ventricular hypertrophy,arteriosclerosis, restenosis, tobacco smoking or peripheral vasculardisease. In some embodiments the peripheral vascular disease isclinically apparent (e.g., peripheral artery disease of Fontaine ClassII or greater). In some embodiments, the risk factor is elevatedtriglycerides, systemic inflammation, high blood phosphorus levels, highparathyroid hormone levels, microalbuminuria, or high homocysteinelevels. In some embodiments, the risk factor is obesity, hyperglycemia,chronic renal failure, high blood glucose, chronic kidney disease, ormetabolic syndrome. In some embodiments, the risk factor is end stagerenal disease. In some embodiments, the risk factor is hypertension,dyslipidemia, hyperlipidemia, elevated total cholesterol, elevated LDLcholesterol, or low HDL cholesterol or atherosclerosis. In someembodiments, the hypertension is manifested as a blood pressure ofgreater than or equal to 180/110 mm Hg. In some other embodiments, thehypertension is mild-to-moderate, with systolic blood pressure (SBP) of140 to 180 mm Hg and/or diastolic blood pressure (DBP) of 90 to 110 mmHg. In some embodiments, the subject has elevated levels of C-reactiveprotein (CRP). In some embodiments, the subject is older than 55 years.In some embodiments, the subject is older than 65 years. In someembodiments, the subject is non-hypertensive. In some embodiments, thesubject has poorly controlled hypertension. In some embodiments, thesubject has a “Type A” personality. In some embodiments, the subject hasa sedentary lifestyle. In some embodiments, the subject has diabetesmellitus. In some embodiments, the diabetes mellitus is Type 2 diabetes.In some embodiments, the subject has a history of two or more said riskfactors. In some embodiments, the subject has a history of three or moresaid risk factors.

In some embodiments, administering said therapeutically effective amountof an anti-IL-1β binding antibody or binding fragment thereof issufficient to achieve a decrease in CRP levels.

The present disclosure also provides methods of reducing mortalityfollowing a cardiovascular event in a subject, comprising administeringto said subject a therapeutically effective amount of an anti-IL-1βbinding antibody or binding fragment thereof. In some embodiments, thecardiovascular event is myocardial infarction, stroke, cardiac arrest,congestive heart failure, cardiovascular death, acute coronary syndrome(e.g., diagnosed), angina or a revascularization procedure. In someembodiments, the cardiovascular event is myocardial infarction or acutecoronary syndrome. In some embodiments, the myocardial infarction ismyocardial infarction with ST elevation (e.g., ST-segment elevationmyocardial infarction, STEMI). In some embodiments, the myocardialinfarction is myocardial infarction without ST elevation (e.g.,non-ST-segment elevation myocardial infarction, NSTEMI). In someembodiments the presence or absence of ST elevation is determined byelectrocardiogram (e.g., ECG, EKG).

In some embodiments, mortality is death from cardiovascular causes. Inother embodiments, mortality is death from any cause. In someembodiments, the cardiovascular event is myocardial infarction, stroke,congestive heart failure, acute coronary syndrome, angina or arevascularization procedure. In some embodiments, the revascularizationprocedure is a coronary, carotid or peripheral arterialrevascularization procedure. In some embodiments, the coronary, carotidor peripheral arterial revascularization procedure is a percutaneouscoronary intervention (PCI), a stent implant, coronary artery bypassgraft (CABG), carotid endarterectomy, peripheral vascular disease bypasssurgery, or peripheral angioplasty surgery.

In some preferred embodiments the therapeutically effective amount of ananti-IL-1β binding antibody or binding fragment thereof is firstadministered within 1 week of the cardiovascular event, within 96 hoursof the cardiovascular event, within 72 hours of the cardiovascularevent, within 48 hours of the cardiovascular event, within 24 hours ofthe cardiovascular event, or within 12 hours of the cardiovascularevent. In some embodiments, the subject does not have Type 2 diabetes.In some embodiments, the subject has survived a previous cardiovascularevent of myocardial infarction or stroke. In some embodiments, theoccurrence of said cardiovascular event is a reoccurrence of acardiovascular event of myocardial infarction or stroke.

In some embodiments, the subject has a history of one or more riskfactors for cardiovascular disease. In some embodiments, the risk factoris manifest coronary heart disease, coronary artery disease, thrombosis,transient ischaemic attack, left ventricular hypertrophy,arteriosclerosis, restenosis, tobacco smoking or peripheral vasculardisease. In some embodiments the peripheral vascular disease isclinically apparent (e.g., peripheral artery disease of Fontaine ClassII or greater). In some embodiments, the risk factor is elevatedtriglycerides, systemic inflammation, high blood phosphorus levels, highparathyroid hormone levels, microalbuminuria, or high homocysteinelevels. In some embodiments, the risk factor is obesity, hyperglycemia,chronic renal failure, high blood glucose, chronic kidney disease, ormetabolic syndrome. In some embodiments, the risk factor ishypertension, dyslipidemia, hyperlipidemia, elevated total cholesterol,elevated LDL cholesterol, or low HDL cholesterol or atherosclerosis. Insome embodiments, the hypertension is manifested as a blood pressure ofgreater than or equal to 180/110 mm Hg. In some other embodiments, thehypertension is mild-to-moderate, with systolic blood pressure (SBP) of140 to 180 mm Hg and/or diastolic blood pressure (DBP) of 90 to 110 mmHg.

In some embodiments, the subject is non-hypertensive. In someembodiments, the subject has poorly controlled hypertension. In someembodiments, the subject has a “Type A” personality. In someembodiments, the subject has a sedentary lifestyle. In some embodiments,the subject has a history of two or more said risk factors. In someembodiments, the subject has a history of three or more said riskfactors.

In some embodiments, administering said therapeutically effective amountof an anti-IL-1β binding antibody or binding fragment thereof issufficient to achieve a decrease in CRP levels.

The present disclosure also provides methods of reducing acardiovascular event in a subject with a history of at least one riskfactor for cardiovascular disease, comprising administering to saidsubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof, and wherein said risk factor isnot Type 2 diabetes, obesity, hyperglycemia, dyslipidemia,hyperlipidemia, chronic renal failure, high blood glucose, chronickidney disease, hypertension, atherosclerosis or metabolic syndrome.

In some embodiments, the cardiovascular event is myocardial infarction,stroke, cardiac arrest, congestive heart failure, cardiovascular death,acute coronary syndrome (e.g., diagnosed), angina or a revascularizationprocedure. In some embodiments, the revascularization procedure is acoronary, carotid or peripheral arterial revascularization procedure. Insome embodiments, the coronary, carotid or peripheral arterialrevascularization procedure is a percutaneous coronary intervention(PCI), a stent implant, coronary artery bypass graft (CABG), carotidendarterectomy, peripheral vascular disease bypass surgery, orperipheral angioplasty surgery.

In some embodiments, the risk factor is manifest coronary heart disease,coronary artery disease, thrombosis, transient ischaemic attack, leftventricular hypertrophy, arteriosclerosis, restenosis, tobacco smokingor peripheral vascular disease. In some embodiments, the risk factor iselevated triglycerides, systemic inflammation, high blood phosphoruslevels, high parathyroid hormone levels, microalbuminuria, or highhomocysteine levels.

In some embodiments, the subject has elevated levels of C-reactiveprotein (CRP). In some embodiments, the subject is older than 55 years.In some embodiments, the subject is older than 65 years. In someembodiments, the subject has a history of two or more said risk factors.In some embodiments, the subject has a history of three or more saidrisk factors.

In some embodiments, administering said therapeutically effective amountof an anti-IL-1βbinding antibody or binding fragment thereof issufficient to achieve a decrease in CRP levels.

The present disclosure also provides methods of treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof and at least one other pharmaceuticalcomposition comprising an active agent other than an IL-1β antibody orfragment.

In some embodiments, the active agent of said at least one otherpharmaceutical composition is a cholesterol lowering agent, a statin, anHMG-CoA reductase inhibitor, a calcium channel blocker, a beta blocker,an antihypertensive, a diuretic, aspirin, niacin, anangiotensin-converting enzyme (ACE) inhibitor, an angiotensin IIreceptor blocker, a vasodilator, an anticoagulant, a inhibitor ofplatelet aggregation, a thrombolytic or digitalis.

The present disclosure also provides methods of treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof and a revascularization procedure. In someembodiments, the revascularization procedure is a coronary, carotid orperipheral arterial revascularization procedure.

The present disclosure also provides methods of reducing restenosis in asubject following a revascularization procedure, comprisingadministering to said subject a therapeutically effective amount of ananti-IL-1β binding antibody or binding fragment thereof. In someembodiments, the revascularization procedure is a coronary, carotid orperipheral arterial revascularization procedure.

The present disclosure also provides methods of treating acutehypertension in a subject comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof and one or more antihypertensive agents. Insome embodiments, the subject has a blood pressure of greater than orequal to 180/110 mm Hg. In some other embodiments, the subject hasmild-to-moderate hypertension, with systolic blood pressure (SBP) of 140to 180 mm Hg and/or diastolic blood pressure (DBP) of 90 to 110 mm Hg.In some embodiments, the antihypertensive agent is administeredintravenously. In some embodiments, the antihypertensive agent isselected from the group consisting of alpha/beta-adrenergic blockingagents, angiotensin-converting enzyme inhibitors, angiotensin IIreceptor antagonists, antiadrenergic agents, beta-adrenergic blockingagents, calcium-channel blocking agents, diuretics, and vasodilators. Insome embodiments, the antihypertensive agent is carvedilol, labetalol,benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril,perindopril, quinapril, ramipril, trandolapril, candesartan, eprosartan,irbesartan, losartan, telmisartan, valsartan, clonidine, doxazosin,guanabenz, guanadrel, guanethidine, guanfacine, mecamylamine,methyldopa, prazosin, reserpine, terazosin, acebutolol, atenolol,betaxolol, bisoprolol, carteolol, metoprolol, nadolol, penbutolol,pindolol, propranolol, timolol, amlodipine, diltiazem, felodipine,isradipine, nicardipine, nifedipine, nisoldipine, verapamil, amiloride,benzthiazide, chlorothiazide, chlorthalidone, furosemide,hydrochlorothiazide, indapamide, metolazone, polythiazide,spironolactone, torsemide, trichlormethiazide, hydralazine,nitroglycerin, sodium nitroprusside, clevidipine or minoxidil. In someembodiments, the antihypertensive agent is labetalol, metoprolol,hydralazine, nitroglycerin, nicardipine, sodium nitroprusside orclevidipine.

The present disclosure also provides methods of reducing, preventing ortreating a cardiovascular event or disease in a subject comprisingadministering to the subject an anti-IL-1β binding antibody or bindingfragment thereof in combination with (e.g., before, during or after) amedical or surgical intervention. Such antibodies may be administered intherapeutically effective amounts. Such interventions may betherapeutically effective. In some embodiments, a medical interventionis an active agent, such as a drug or a biologic, including, forexample, any one or more of the active agents described herein. In someembodiments, a medical intervention is an out-patient medical treatmentor procedure. In some embodiments, a medical intervention is anin-patient hospitalization. In some embodiments, a surgical interventionis a revascularization procedure, including, for example, any one ormore of the revascularization procedures described herein. In someembodiments, a surgical intervention involves a heart valve repair orreplacement, coronary bypass surgery, heart transplant or heart pump. Insome embodiments, a surgical intervention involves a biventricularcardiac pacemaker, internal cardiac defibrillator (ICD) or myectomy. Insome embodiments, a medical intervention is smoking cessation medicationor smoking cessation counseling.

The present disclosure also provides methods of reducing acardiovascular event in a subject with a history of at least one riskfactor for cardiovascular disease, comprising (a) identifying,diagnosing or selecting the subject with the history of at least onerisk factor for cardiovascular disease and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof, and wherein the cardiovascularevent is myocardial infarction, stroke, cardiovascular death, congestiveheart failure, cardiac arrest, acute coronary syndrome, angina, or arevascularization procedure.

The present disclosure also provides methods of reducing acardiovascular event in a subject with a history of a previouscardiovascular event, comprising (a) identifying, diagnosing orselecting the subject with the history of the previous cardiovascularevent and (b) administering to the subject a therapeutically effectiveamount of an anti-IL-1β binding antibody or binding fragment thereof,and wherein the cardiovascular event is myocardial infarction, stroke,acute coronary syndrome, angina or a revascularization procedure.

The present disclosure also provides methods of reducing mortalityfollowing a cardiovascular event in a subject, comprising (a)identifying, diagnosing or selecting the subject having thecardiovascular event and (b) administering to the subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof.

The present disclosure also provides methods of reducing acardiovascular event in a subject with a history of at least one riskfactor for cardiovascular disease, comprising (a) identifying,diagnosing or selecting the subject with the history of at least onerisk factor for cardiovascular disease and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof, and wherein the risk factor is notType 2 diabetes, obesity, hyperglycemia, dyslipidemia, hyperlipidemia,chronic renal failure, high blood glucose, chronic kidney disease,hypertension, atherosclerosis or metabolic syndrome.

The present disclosure also provides methods of treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising (a) identifying, diagnosing or selectingthe subject with the cardiovascular event and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof and at least one otherpharmaceutical composition comprising an active agent other than anIL-1β antibody or fragment.

The present disclosure also provides methods for treating acardiovascular event in a subject, wherein the cardiovascular event ismyocardial infarction, stroke, congestive heart failure, acute coronarysyndrome or angina, comprising (a) identifying, diagnosing or selectingthe subject with the cardiovascular event and (b) administering to thesubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof and a revascularization procedure.

The present disclosure also provides methods of reducing restenosis in asubject following a revascularization procedure, comprising (a)identifying, diagnosing or selecting the subject with therevascularization procedure and (b) administering to the subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof.

The present disclosure also provides methods of treating acutehypertension in a subject comprising (a) identifying, diagnosing orselecting the subject with acute hypertension and (b) administering tothe subject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof and one or more antihypertensiveagents. In some embodiments, the hypertension is manifested as a bloodpressure of greater than or equal to 180/110 mm Hg. In some otherembodiments, the hypertension is mild-to-moderate, with systolic bloodpressure (SBP) of 140 to 180 mm Hg and/or diastolic blood pressure (DBP)of 90 to 110 mm Hg.

The present disclosure also provides methods of reducing in a subjectwith a history of recent myocardial infarction (MI), recent stroke, orestablished peripheral arterial disease, the rate of a combined endpointof new ischemic stroke (fatal or not), new MI (fatal or not), and othervascular death, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof. In some embodiments, the subject has acutecoronary syndrome without ST segment elevation (e.g., unstable angina ornon-Q-wave myocardial infarction). In some embodiments, the anti-IL-1βbinding antibody or binding fragment thereof is administered initiallywithin 12, 24, 48 or 72 hours of onset of the most recent episode ofchest pain or symptoms consistent with ischemia. In some embodiments,the subject has either ECG changes compatible with new ischemia (e.g.,without ST segment elevation) or elevated cardiac enzymes or troponin Ior T to at least twice the upper limit of normal.

The present disclosure also provides methods of reducingatherothrombotic events in a subject with a history of recent myocardialinfarction (MI), recent stroke, or established peripheral arterialdisease, comprising administering to said subject a therapeuticallyeffective amount of an anti-IL-1β binding antibody or binding fragmentthereof. In some embodiments, the subject has acute coronary syndromewithout ST segment elevation (e.g., unstable angina or non-Q-wavemyocardial infarction). In some embodiments, the anti-IL-1β bindingantibody or binding fragment thereof is administered initially within 72hrs, or preferably 48 hours, or more preferably 24, 12, 6 or 3 hours ofonset of the most recent episode of chest pain or symptoms consistentwith ischemia. In some embodiments, the subject has either ECG changescompatible with new ischemia (e.g., without ST segment elevation) orelevated cardiac enzymes or troponin I or T to at least twice the upperlimit of normal.

The present disclosure also provides methods of reducing in subjectswith ST-segment elevation acute myocardial infarction, the rate of deathfrom any cause and the rate of a combined endpoint of death,re-infarction or stroke, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof. In some embodiments, the anti-IL-1β bindingantibody or binding fragment thereof is administered initially within 72hrs, or preferably 48 hours, or more preferably 24, 12, 6 or 3 hours ofthe subject presenting with symptoms of myocardial infarction.

In some embodiments of any of the methods described above, the subjectis a patient with cardiovascular disease, including, for example, acutecardiovascular disease or chronic cardiovascular disease.

In some embodiments of any of the methods described above, administeringsaid therapeutically effective amount of an anti-IL-1β binding antibodyor binding fragment thereof is sufficient to achieve a decrease in CRPlevels.

The present disclosure also provides uses of an anti-IL-1β bindingantibody or binding fragment thereof which has a lower IC₅₀ than anIL-1β receptor antagonist in a human whole blood IL-1β inhibition assaythat measures IL-1β induced production of IL-8, in the manufacture of acomposition for use in the reduction, prevention or treatment of acardiac event or a cardiovascular disease.

The disclosure also provides that a reduction of a cardiovascular event(e.g., delaying time to event, reducing likelihood or risk of event,preventing an event, reducing severity of event, reducing time torecovery) may be evaluated in subjects over a period of 2 or more years,3 or more years, 4 or more years, or 5 or more years following thecardiovascular event and/or initial administration of the IL-1β bindingantibody or binding fragment thereof.

In addition, the disclosure further provides that a therapeuticallyeffective amount of anti-IL-1β binding antibody or binding fragmentthereof may also be sufficient to achieve a decrease in C-reactiveprotein (CRP) levels. The reduction in CRP levels is readily measuredusing standard assays (e.g., high-sensitivity CRP, ultra-sensitive CRP).As provided by the methods disclosed herein, the decrease in C-reactiveprotein levels may, for example, be a decrease of ≧0.2, ≧0.4, ≧0.6,≧0.8, ≧1.0, ≧1.4, ≧1.8, ≧2.2, ≧2.6, ≧3.0 mg/L from pre-treatment levels.Alternatively, the decrease in C-reactive protein levels may, forexample, be a decreaseof >20%, >30%, >40%, >50%, >60%, >70%, >80%, >90%, >95% frompre-treatment levels.

Alternatively, or in addition, subjects treated as disclosed herein mayexperience a measurable improvement in lipid profile (e.g., decrease inserum lipids, change in ratio of HDL and LDL). Such measurements ofserum lipids and/or lipid profile may include, for example a decrease incholesterol, a decrease in low-density lipoprotein cholesterol (LDL), adecrease in very-low-density lipoprotein cholesterol (VLDL), a decreasein triglycerides, a decrease in free fatty acids, a decrease inapolipoprotein B (Apo B), an increase in high-density lipoproteincholesterol (HDL), maintaining the level of high-density lipoproteincholesterol (HDL) compared to pre-treatment level, and/or an increase inapolipoprotein A (Apo A). Measurements may be using standard techniquesknown in the art. For example, a decrease in the level of cholesterol(e.g., total cholesterol) may be a decrease of at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more from thepre-treatment level. A decrease in the level of low-density lipoproteincholesterol may be a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, or more from the pre-treatment level. Adecrease in the triglyceride level in the blood of the subject may be adecrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, or more from the pre-treatment level. A decrease inthe level of free fatty acids may be a decrease of at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more from thepre-treatment level. An increase in the level of high-densitylipoprotein cholesterol may be an increase of at least 1%, 2%, 3%, 4%,5%, 6%, 8%, 10%, 12%, 14%, 16%, or more from the pre-treatment level.

The aforementioned diagnoses and measurements may be made using standardmedical practices known in the art and/or any of a variety of standardassays known in the art, such as for example assays published inChemecky C C, Berger B J, eds. (2004). Laboratory Tests and DiagnosticProcedures, 4th ed. Philadelphia: Saunders; Fischbach F T, Dunning M BIII, eds. (2004). Manual of Laboratory and Diagnostic Tests, 7th ed.Philadelphia: Lippincott Williams and Wilkins; Genest J, et al. (2003).Recommendations for the management of dyslipidemia and the prevention ofcardiovascular disease: Summary of the 2003 update. Canadian MedicalAssociation Journal, 169(9): 921-924. Also available online:http://www.cmaj.ca/cgi/content/full/169/9/921/DC1; Handbook ofDiagnostic Tests (2003). 3rd ed. Philadelphia: Lippincott Williams andWilkins; and Pagana K D, Pagana T J (2002). Mosby's Manual of Diagnosticand Laboratory Tests, 2nd ed. St. Louis: Mosby.

Dosing

Anti-IL-1β binding antibodies or binding fragments thereof for use inany and/or all of the aforementioned methods may be administered in oneor more doses (e.g., initial dose and one or more subsequent doses). Insome embodiments, the anti-IL-1β binding antibody or binding fragmentthereof is administered in one or more doses of 10 mg/kg or less, 5mg/kg or less, 3 mg/kg or less, or 2 mg/kg or less of antibody orfragment. In some embodiments, the anti-IL-1β binding antibody orbinding fragment thereof is administered in one or more doses of 1 mg/kgor less, one or more doses of 0.5 mg/kg or less, one or more doses of0.3 mg/kg or less, one or more doses of 0.1 mg/kg or less, or one ormore doses of 0.03 mg/kg or less of antibody or fragment. In some of theaforementioned embodiments, the one or more doses are at least 0.01mg/kg of anti-IL-1β binding antibody or binding fragment thereof. Insome embodiments, the anti-IL-1β binding antibody or binding fragmentthereof is administered in one or more doses of about 0.01 mg/kg to 1mg/kg, about 0.03 mg/kg to 1 mg/kg, about 0.01 mg/kg to 0.3 mg/kg, orabout 0.1 mg/kg to 0.3 mg/kg. In some embodiments, the anti-IL-1βbinding antibody or binding fragment thereof is administered in one ormore doses of about 0.001 mg/kg to 0.3 mg/kg, about 0.001 mg/kg to 0.1mg/kg, about 0.001 mg/kg to 0.03 mg/kg or about 0.001 mg/kg to 0.01mg/kg.

In other embodiments, the initial dose and one or more subsequent dosesof anti-IL-1β binding antibody or binding fragment thereof are each fromabout 0.01 mg/kg to about 10 mg/kg of antibody, from about 0.05 to about5 mg/kg of antibody, from about 0.05 mg/kg to about 3 mg/kg of antibody,from about 0.1 mg/kg to about 3 mg/kg of antibody, from about 0.1 mg/kgto about 1 mg/kg of antibody, from about 0.1 mg/kg to about 0.5 mg/kg ofantibody, from about 0.3 mg/kg to about 5 mg/kg of antibody, from about0.3 mg/kg to about 3 mg/kg of antibody, from about 0.3 mg/kg to about 1mg/kg of antibody, from about 0.5 mg/kg to about 5 mg/kg of antibody,from about 0.5 mg/kg to about 3 mg/kg of antibody, from about 0.5 mg/kgto about 1 mg/kg of antibody, from about 1 mg/kg to about 5 mg/kg ofantibody, or from about 1 mg/kg to about 3 mg/kg of antibody. In certainembodiments, two or more, three or more, four or more, five or more, sixor more, seven or more, eight or more, nine or more, ten or more oreleven or more subsequent doses of the antibody are administered. Theaforementioned dosage amounts refer to mg (antibody or fragment)/kg(weight of the individual to be treated).

Anti-IL-1β binding antibodies or binding fragment thereof for use in anyand/or all of the aforementioned methods may be administered as a fixeddose, independent of a dose per subject weight ratio. In someembodiments, the anti-IL-1β binding antibody or binding fragment thereofis administered in one or more fixed doses of 1000 mg or less, 500 mg orless, or 250 mg or less of antibody or fragment. In some embodiments,the anti-IL-1β binding antibody or binding fragment thereof isadministered in one or more fixed doses of 100 mg or less, 25 mg orless, or 10 mg or less of antibody or fragment. In some embodiments, theanti-IL-1β binding antibody or binding fragment thereof is administeredin one or more doses of at least 0.5 mg of antibody or fragment. In someembodiments, the anti-IL-1β binding antibody or binding fragment thereofis administered in one or more doses of at least 1 mg of antibody orfragment. In some embodiments, the anti-IL-1β binding antibody orbinding fragment thereof is administered in one or more doses of atleast 10 mg of antibody or fragment. In some embodiments, the anti-IL-1βbinding antibody or binding fragment thereof is administered in one ormore doses of 1 mg to 100 mg of antibody or fragment.

In certain embodiments, the fixed dose of anti-IL-1β binding antibody orbinding fragment thereof is from about 1 mg to about 10 mg, about 1 mgto about 25 mg, about 10 mg to about 25 mg, about 10 mg to about 50 mg,about 10 mg to about 100 mg, about 25 mg to about 50 mg, about 25 mg toabout 100 mg, about 50 mg to about 100 mg, about 50 mg to about 150 mg,about 100 mg to about 150 mg, about 100 mg to about 200 mg, about 150 mgto about 200 mg, about 150 mg to about 250 mg, about 200 mg to about 250mg, about 200 mg to about 300 mg, about 250 mg to about 300 mg, about250 mg to about 500 mg, about 300 mg to about 400 mg, about 400 mg toabout 500 mg, about 400 mg to about 600 mg, about 500 mg to about 750mg, about 600 mg to about 750 mg, about 700 mg to about 800 mg, or about750 mg to about 1000 mg. In other embodiments, the fixed dose anti-IL-1βbinding antibody or binding fragment thereof is from about 1 mg to about10 mg, about 1 mg to about 25 mg, about 10 mg to about 25 mg, about 10mg to about 100 mg, about 25 mg to about 50 mg, about 50 mg to about 100mg, about 100 mg to about 150 mg, about 150 mg to about 200 mg, or about200 mg to about 250 mg.

In some embodiments of any and/or all of the aforementioned methods, thefixed dose of anti-IL-1β binding antibody or binding fragment thereof isadministered using a pre-filled syringe or delivery device.

In some embodiments of any and/or all of the aforementioned methods, theanti-IL-1β binding antibody or binding fragment thereof is administeredby subcutaneous, intravenous or intramuscular injection.

In some embodiments of any and/or all of the aforementioned methods,administration of an initial dose of anti-IL-1β binding antibody orbinding fragment thereof is followed by the administration of one ormore subsequent doses. In some embodiments, the initial dose and one ormore subsequent doses are administered at an interval of about onceevery week to about once every 12 months. In some embodiments, theinitial dose and one or more subsequent doses are administered at aninterval of about once every two weeks to about once every 6 months. Insome embodiments, the initial dose and one or more subsequent doses areadministered at an interval of about once every month to about onceevery 6 months. In some embodiments, the initial dose and one or moresubsequent doses are administered at an interval of about once everymonth to about once every 3 months. In some embodiments, the initialdose and one or more subsequent doses are administered at an interval ofabout once every 3 months to about once every 6 months.

The disclosure also provides dosing regimens for use in any and/or allof the aforementioned methods, wherein the dosing regimens comprise morethan one dosing interval for administration of an IL-1β binding antibodyor binding fragment thereof. In some embodiments, the dosage regimencomprises at least two (e.g., two, three, four, five, six) differentdosing intervals for administration of the IL-1β antibody or fragmentthereof. In some embodiments, the dosage regimen comprises two differentdosing intervals for administration of the IL-1β antibody or fragmentthereof. In some embodiments, the dosing regimen comprises two differentdosing intervals for administration of the IL-1β binding antibody orbinding fragment thereof, wherein a first dosing interval comprisesadministration of one or more doses of the IL-1β antibody or fragmentthereof and a second dosing interval comprises administration of one ormore doses of the IL-1β antibody or fragment thereof, and wherein thefirst dosing interval is shorter in time than the second dosinginterval. For example, the first dosing interval may be days or weeks,and the second dosing interval may be months. In some embodiments, thefirst dosing interval is about 5 days to about 28 days, about 7 days toabout 21 days, about 12 days to about 16 days, or about 14 days. In someembodiments, the second dosing interval is about 1 month to about 3months, about 1 month to about 2 months, or about 1 month. In someembodiments, the first dosing interval is about 7 days and the seconddosing interval is about 1 month.

In some embodiments, administration of an initial dose of anti-IL-1βbinding antibody or binding fragment thereof is followed byadministration of one or more subsequent doses, and wherein the dosingintervals between administration of the initial dose and a second dose,and the second dose and a third dose are about 7 days to about 21 days,and wherein the dosing intervals between administration of subsequentdoses is about 1 month to about 3 months. In some embodiments, thedosing intervals between administration of the initial dose and a seconddose, and the second dose and a third dose are about 12 to 16 days, andthe dosing intervals between administration of subsequent doses is about1 month to about 2 months. In some embodiments, the dosing intervalsbetween administration of the initial dose and a second dose, and thesecond dose and a third dose are about 14 days, and the dosing intervalsbetween administration of subsequent doses is about 1 month. In someembodiments of any and/or all of the aforementioned methods, theanti-IL-1β binding antibody or binding fragment thereof is administeredto a subject such that the interval between doses is a time sufficientto maintain a plasma concentration of said antibody or antibody fragmentin the subject at a level of at least about 0.1 ug/mL. In someembodiments, the anti-IL-1β binding antibody or binding fragment thereofis administered to a subject such that the interval between doses is atime sufficient to maintain a plasma concentration of said antibody orantibody fragment in the subject at a level of at least about 0.3 ug/mL.In some embodiments, the anti-IL-1β binding antibody or binding fragmentthereof is administered to a subject such that the interval betweendoses is a time sufficient to maintain a plasma concentration of saidantibody or antibody fragment in the subject at a level of at leastabout 1 ug/mL. In some embodiments, these plasma concentration valuesrefer to values obtained for an individual that is treated with theantibody of fragment in accordance with the disclosure herein.

In some embodiments of any and/or all of the aforementioned methods,administration of an initial dose of the anti-IL-1β binding antibody orbinding fragment thereof is followed by the administration of one ormore subsequent doses, and wherein said one or more subsequent doses arein an amount that is approximately the same or less than the initialdose.

In some embodiments of any and/or all of the aforementioned methods,administration of an initial dose of the anti-IL-1β binding antibody orbinding fragment thereof is followed by the administration of one ormore subsequent doses, and wherein at least one of the subsequent dosesis in an amount that is more than the initial dose.

In some embodiments of any and/or all of the aforementioned methods, theanti-IL-1β binding antibody or binding fragment thereof has a lower IC₅₀than an IL-1β receptor antagonist in a human whole blood IL-1βinhibition assay that measures IL-1β induced production of IL-8. In someembodiments, the IL-1β receptor antagonist is anakinra.

In some embodiments of any and/or all of the aforementioned methods, ananti-IL-1β binding antibody or binding fragment is administered, whereinadministration of an initial dose of the antibody or antibody fragmentis followed by the administration of one or more subsequent doses, andwherein the plasma concentration of said antibody or antibody fragmentin the human is permitted to decrease below a level of about 0.1 ug/mLfor a period of time greater than about 1 week and less than about 6months between administrations during a course of treatment with saidinitial dose and one or more subsequent doses. In some embodiments, theplasma concentration of said antibody or antibody fragment is permittedto decrease below a level of about 0.07 ug/mL, about 0.05 ug/mL, about0.03 ug/mL or about 0.01 ug/mL for a period of time greater than about 1week and less than about 5 months, about 4 months, about 3 months, about2 months, about 1 month, about 3 weeks, or about 2 weeks betweenadministrations. In some embodiments, the plasma concentration valuesrefer to values obtained for an individual that is treated with theantibody of fragment in accordance with the disclosure herein.

Combinations

The disclosure also provides that pharmaceutical compositions comprisingone or more other active agents may be administered in conjunction with(e.g., separately from) the IL-1β binding antibodies or fragments, andsuch administrations may be performed at the same point or differentpoints in time, such as for example the same or different days.Administration of the other active agents may be according to standardmedical practices known in the art (e.g., current standard of care), orthe administration may be modified (e.g., longer intervals, smallerdosages, delayed initiation) when used in conjunction withadministration of IL-1β binding antibodies or fragments, such asdisclosed herein. The active agents set forth below are exemplary andnot intended to be limiting. combinations can also include more than oneadditional agent, e.g., two or three additional agents.

Anti-IL-1β antibodies or fragments thereof administered to a subject inas disclosed herein may be administered in combination with treatmentwith at least one additional active agent, such as for example any ofthe active agents provided herein. In one embodiment, treatment with theat least one active agent is maintained. In another embodiment,treatment with the at least one active agent is reduced or discontinued(e.g., when the subject is stable), while treatment with the anti-IL-1βantibody or fragment is maintained at a constant dosing regimen. Inanother embodiment, treatment with the at least one active agent isreduced or discontinued (e.g., when the subject is stable), andtreatment with the anti-IL-1β antibody or fragment is reduced (e.g.,lower dose, less frequent dosing, shorter treatment regimen). In anotherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), and treatment with theanti-IL-1β antibody or fragment is increased (e.g., higher dose, morefrequent dosing, longer treatment regimen). In yet another embodiment,treatment with the at least one active agent is maintained and treatmentwith the anti-IL-1β antibody or fragment is reduced or discontinued(e.g., lower dose, less frequent dosing, shorter treatment regimen). Inyet another embodiment, treatment with the at least one active agent andtreatment with the anti-IL-1β antibody or fragment are reduced ordiscontinued (e.g., lower dose, less frequent dosing, shorter treatmentregimen).

In some embodiments, any of the methods described above may furthercomprise administering at least one other pharmaceutical compositioncomprising an active agent other than an anti-IL-1β binding antibody orbinding fragment thereof. In some embodiments, the active agent of saidat least one other pharmaceutical composition is a cholesterol loweringagent. In some embodiments, the active agent of said at least one otherpharmaceutical composition is a statin or an HMG-CoA reductase inhibitor(e.g., lovastatin, pravastatin, simvastatin, fluvastatin, atorvastatin,cerivastatin, mevastatin, pitavastatin, rosuvastatin or mixtures thereofor mixtures with Ezetimibe, niacin, Amlodipine Besylate). In someembodiments, the active agent of said at least one other pharmaceuticalcomposition is a calcium channel blocker (e.g., amlodipine, diltiazem,nifedipine, nicardipine, verapamil) or a beta blocker (e.g., esmolol,metoprolol, nadolol, penbutolol). In some embodiments, the active agentof said at least one other pharmaceutical composition is anantihypertensive (e.g., labetalol, metoprolol, hydralazine,nitroglycerin, nicardipine, sodium nitroprusside, clevidipine), adiuretic (e.g., a thiazide diuretic, chlorthalidone, furosemide,hydrochlorothiazide, indapamide, metolazone, amiloride hydrochloride,spironolactone, triamterene) or aspirin. In some embodiments, the activeagent of said at least one other pharmaceutical composition is anangiotensin-converting enzyme (ACE) inhibitor (e.g. ramipril,ramiprilat, captopril, lisinopril) or an angiotensin II receptor blocker(e.g., losartan, olmesartan, valsartan). In some embodiments, the activeagent of said at least one other pharmaceutical composition is avasodilator. In some embodiments, the active agent of said at least oneother pharmaceutical composition is an anticoagulant (e.g.,acenocoumarol, phenprocoumon, warfarin heparin, low molecular weightheparin) or inhibitor of platelet aggregation (e.g., clopidogrel,ticlopidine, cilostazol, dipyridamole, eptifibatide, aspirin, abciximab,eptifibatide, tirofiban). In some embodiments, the active agent of saidat least one other pharmaceutical composition is a thrombolytic (e.g.,streptokinase, urokinase, alteplase, reteplase, tenecteplase). In someembodiments, the active agent of said at least one other pharmaceuticalcomposition is digitalis. In some embodiments, the active agent of saidat least one other pharmaceutical composition is digoxin or nesiritide.In some embodiments, the active agent of said at least one otherpharmaceutical composition is oxygen. In some embodiments, the activeagent of said at least one other pharmaceutical composition is athrombin inhibitor (e.g., hirudin, bivalirudin). In some embodiments,the active agent of said at least one other pharmaceutical compositionis a nitrate (e.g., glyceryl trinitrate (GTN)/nitroglycerin, isosorbidedinitrate, isosorbide mononitrate). In some embodiments, the activeagent of said at least one other pharmaceutical composition is ananalgesic (e.g., morphine sulfate). In some embodiments, the activeagent of said at least one other pharmaceutical composition is a renininhibitor. In some embodiments, the active agent of said at least oneother pharmaceutical composition is an endothelin A receptor inhibitor.In some embodiments, the active agent of said at least one otherpharmaceutical composition is an aldosterone inhibitor.

In another embodiment, the use of the IL-1β antibodies or bindingfragments is contemplated in the manufacture of a medicament fortreating or preventing a disease or condition as disclosed herein (e.g.,for the reduction, prevention or treatment of cardiovascular eventsand/or cardiovascular diseases). In any of the uses, the medicament canbe coordinated with treatment using a second active agent.

In yet another aspect of the present disclosure, an article ofmanufacture is provided, comprising a container, a composition withinthe container comprising an anti-IL-1β antibody or fragment thereof, anda package insert containing instructions to administer the antibody orfragment to a subject (e.g., human) as disclosed herein (e.g., for thereduction, prevention or treatment of cardiovascular events and/orcardiovascular diseases). In one embodiment, the container furthercomprises a pharmaceutically suitable carrier, excipient or diluent. Ina related embodiment, the composition within the container furthercomprises a second active agent.

Kits are also contemplated by the disclosure. In one embodiment, a kitcomprises a therapeutically or prophylactically effective amount of ananti-IL-1β antibody or fragment thereof, packaged in a container, suchas a vial or bottle, and further comprising a label attached to orpackaged with the container, the label describing the contents of thecontainer and providing indications and/or instructions regarding use ofthe contents of the container as disclosed herein (e.g., for thereduction, prevention or treatment of cardiovascular events and/orcardiovascular diseases). In one embodiment, the container furthercomprises a pharmaceutically suitable carrier, excipient or diluent. Ina related embodiment, the container further contains a second activeagent.

In one embodiment, the article of manufacture, kit or medicament is forthe treatment or prevention of a disease or condition in a subject(e.g., human) as disclosed herein (e.g., for the reduction, preventionor treatment of cardiovascular events and/or cardiovascular diseases).In another embodiment, the instructions of a package insert of anarticle of manufacture or label of a kit comprise instructions foradministration of the antibody or fragment according to any of theaforementioned dose amounts and/or dosing regiments. In yet anotherembodiment, the container of kit or article of manufacture is apre-filled syringe.

EXAMPLES

The following examples are intended merely to further illustrate thepractice of the present disclosure, but should not be construed as inany way limiting its scope. The disclosures of all patent and scientificliteratures cited within are hereby expressly incorporated in theirentirety by reference.

Example 1 Administration of an IL-1β Antibody to Human Subjects

IL-1β binding antibodies or binding fragments thereof may beadministered to a subject for the aforementioned uses. Specifically, inone example, an IL-1β antibody designated AB7 (described above) wasadministered to human subjects to evaluate safety, pharmacokinetics, andin vivo biological activity. A double-blind, placebo controlled clinicalstudy was performed in human subjects with Type 2 diabetes. Groups ofsubjects were given the antibody by either the intravenous (IV) orsubcutaneous (SC) route, and either as a single doses or as multipledoses over a period of time.

The treatment groups and numbers of subjects for the study are shown inthe following table for a single dose by the IV route of administration.

IV Route Antibody Placebo Group # Subjects Dose # Subjects 1 5 0.01mg/kg 1 2 5 0.03 mg/kg 1 3 5 0.1 mg/kg 1 4 5 0.3 mg/kg 1 5 5 1.0 mg/kg 16 5 3.0 mg/kg 1

Similarly, treatment groups and numbers of subjects are shown in thefollowing table for single and multiple (3 times, biweekly) doses by theSC route of administration.

SC Route Antibody Placebo Group # Subjects Dose # Subjects Single Dose 15 0.03 mg/kg 1 2 5 0.1 mg/kg 1 3 5 0.3 mg/kg 1 Multi Dose 4 5 0.03 mg/kg1 5 5 0.3 mg/kg 1

On study Day 1, antibody or placebo was administered via constant rateIV infusion or SC injection (e.g., anterior abdomen, arm, thigh). Safetyassessments, including the recording of adverse events, physicalexaminations, vital signs, and clinical laboratory tests (e.g., bloodchemistry, hematology, urinalysis) were conducted using standard medicalpractices known in the art. Blood samples were collected pre-doseadministration and at multiple time periods post-administration toassess various parameters, including C-reactive protein.

Alternatively or in addition, study groups also may be included toevaluate, for example, the administration of additional numbers ofsubsequent doses at the same or longer intervals (e.g., monthlyinterval), alternative dose amounts, and/or increased group sizes.

Example 2 Pharmacokinetics of an IL-1β Antibody in Human Subjects

Samples are obtained for pharmacokinetic analysis at days 0, 1, 2, 3, 4,7, 9±1, 11±1, 14±1, 21±2, 28±2, 42±3, and 56±3. Interim analysis ofpharmacokinetic data following IV administration of a single dose ofantibody at the 0.01, 0.03, 0.1, 0.3, or 1.0 mg/kg dose levels showedserum concentration-time profiles with a terminal half-life of 22 days,clearance of 2.54 mL/day/kg and volume of distribution of the centralcompartment of 41.3 mL/kg, very similar to serum volume (FIG. 1).

Similarly, samples were analyzed for the single dose SC administrationgroups. As shown in FIG. 2, administration of the antibody at 0.03, 0.1and 0.3 mg/kg dose levels yielded profiles with a terminal half-life of22.7 days, clearance of 2.4 mL/day/kg and volume distribution of thecentral compartment of 40.7 mL/kg.

Example 3 Effect of an IL-1β Antibody on CRP in Human Subjects

C-reactive protein also was measured in serum at the same time points asthe PK samples. A single dose of antibody reduced ultrasensitiveC-reactive protein (usCRP) levels in each of the antibody treatment dosegroups compared to placebo. As shown in FIG. 3, at 28 days after asingle IV dose of antibody, the median percent reductions in usCRP were33, 46, 47, 36, and 26 for the 0.01, 0.03, 0.1, 0.3, and 1.0 mg/kg dosegroups, respectively, compared to 4 percent for placebo.

Example 4 Evaluation of an IL-1β Antibody in a Cardiovascular EventModel (Acute Myocardial Infarction)

To determine the cardioprotective effect (e.g., inhibiting adversecardiac remodeling) of an IL-1β antibody or binding fragment thereof, arodent model of acute myocardial infarction (MI) may be used (see forexample, Wang et al., 2006, Tex. Heart Inst. J. 33:290-293; Salloum etal., 2009, Cardiovasc. Drugs Ther. 23:129-135). Improvements inmeasurements of heart function, such as for example in the MI model, arerelated to the chance of a subsequent cardiovascular event (e.g.,congestive heart failure). Outbred mice (e.g., Institute of CancerResearch mice) and/or rats (e.g., Wistar rats) are used in the rodent MImodel. Prior to surgery, the animals are evaluated by transthoracicechocardiography (TTE), for example, using a Vevo770 imaging system(VisualSonics, Toronto, Canada) or Acuson C256, to obtain measurementsfor the following parameters:

-   -   Left ventricular end-diastolic diameter (LVEDD)    -   Left ventricular end-systolic diameter (LVESD)    -   Anterior wall diastolic thickness (AWDT)    -   Posterior wall diastolic thickness (PWDT)    -   Anterior wall systolic thickness (AWST)    -   Posterior wall systolic thickness (PWST)        Left ventricular fractional-shortening (FS) is calculated as:

(LVEDD−LVESD)/LVEDD×100

Adult animals under anesthesia are subjected to coronary arteryligation. After thoracotomy to expose the heart, MI is induced byligation of the proximal left descending coronary artery using a silkligature placed around the vessel. Control animals (sham operation) aresubjected to the same surgical procedure, but without the coronaryligation (see following table). Animals that die during or immediatelyafter the postoperative period are not included in the analyses.

TTE Surgery Antibody Txt Repeat TTE Group 1 Yes Yes, High dose (t = 0)24 hr, 7 d, MI induction 14 d Group 2 Yes Yes, Low dose (t = 0) 24 hr, 7d, MI induction 14 d Group 3 Yes Yes, High dose (24 hr) 24 hr, 7 d, MIinduction 14 d Group 4 Yes Yes, Low dose (24 hr) 24 hr, 7 d, MIinduction 14 d Group 3 Yes Yes, Placebo 24 hr, 7 d, MI induction 14 dGroup 4 Yes Yes, Sham N/A 24 hr, 7 d, operation 14 d

Animals then receive either the treatment antibody or placebo (e.g.,control antibody) administered intraperitoneally or intravenously at oneor more pre-determined times during and/or following ischemia. Forexample, in one group the antibody is administered during ischemia (t=0)and in another group, the antibody is administered 24 hours afterischemia.

The animals are observed and numbers of deaths during the study periodare recorded. The remaining animals again are evaluated by TTE atpre-determined post-treatment days (e.g., 24 hr, Day 7, Day 14).Systolic B P also may be measured in conscious awake awake, for example,using a noninvasive computerized tail-cuff system (BP-2000, VisitechSystems), which has been found to correlate closely with directintraarterial measurement of BP. Animals are sacrificed, blood collectedfor serum, and the infarct area (size) determined. After removal, theheart is subjected to staining with Evans blue dye or 0.5% nitrobluetetrazolium (NBT), rinsed with saline and photographed to determineinfarct size. The tissue is then fixed in 4% paraformaldehyde, embeddedin paraffin and sectioned for staining with hematoxylin and eosin forhistologic evaluation of tissue damage. Alternatively or additionally,tissue is fixed and sectioned to quantitate the level of cardiomyocytecell death (e.g., TUNEL to determine apoptosis).

Alternatively, studies to evaluate the effect of an IL-1β antibody orfragment on heart function and/or adverse cardiac remodeling (e.g.,chance of a subsequent cardiovascular event, such as for example,congestive heart failure) may be performed in adult male out-bred ICRmice (e.g., Harlan Laboratories (Indianapolis, Ind.)). CD-1 miceunderwent experimental myocardial infarction as previously described(Abbate et al., 2008, Circulation 117:2670-2683). Mice were anesthetizedwith pentobarbital (70 mg/kg, IP), intubated orotracheally, andventilated on a positive-pressure ventilator. Left thoracotomy wasperformed at the fourth intercostal space and the heart was exposed bystripping the pericardium. The left descending coronary artery was thenidentified with a surgical microscope (Leica F40) and ligated with a 7.0silk ligature. A group of 4 mice underwent sham operation as previouslydescribed (Abbate, ibid). After surgery, mice were randomly assigned totreatment with the anti-IL-1β antibody XMA052 MG1K, administeredintraperitoneally (0.05 mg/kg, 0.5 mg/kg, 5 mg/kg doses) or a controlIgG (n=6 per group) immediately after surgery and then again 7 dayslater. The effect of pretreatment with an additional dose of theantibody (0.5 mg/kg) 48 hours prior to surgery also was tested.

All mice underwent transthoracic echocardiography before surgery and at7, 14 and 28 days after coronary ligation. Doppler echocardiography wasperformed with the Vevo770 imaging system (VisualSonics Inc, Toronto,Ontario, Canada) and a 30-MHz probe. The heart was first imaged in the2-dimensional mode in the parasternal and apical views and measurementswere performed according to the to the American Society ofEchocardiography recommendations (Gardin et al., 2002, J Am SocEchocardiography 15:275-290). The left ventricular (LV) end-diastolicdiameter (LVEDD), LV end-systolic diameters (LVESD), anterior walldiastolic thickness (AWDT), anterior wall systolic thickness (AWST),posterior wall diastolic thickness (PWDT), and posterior wall systolicthickness (PWST) were measured at M-mode. LV fractional shortening(LVFS) was calculated as follows: FS=(LVEDD-LVESD)/LVEDDx100. The numberof akinetic segments (which correlates with infarct size) was determinedusing a 17-segment map. An apical view was used to measure the ejectiontime (ET), the time interval between the end of the transmitral A waveand the following E wave (AE). The myocardial performance index (MPI, orTei index) was then computed (MPI=[AE−ET]/ET). The tricuspidal annularplane systolic excursion was also measured as a marker of rightventricular function. The investigator performing and reading theechocardiogram was blinded to the treatment allocation. The SPSS 11.0(Chicago, Ill.) was used for the statistical analysis, using ANOVA formultiple comparisons with post-hoc T-test to explore between groupdifferences. For comparisons of interval changes between multiplegroups, random effects ANOVA for repeated-measures was used to determinethe main effect of time, group, and time-by-group interaction.Statistical differences were considered significant if the P value was<0.05.

Baseline echocardiographic values were similar in all groups. Asexpected, significant increases in LV diameters (LVEDD and LVESD) and asignificant decrease in LVFS were observed as early as 7 days aftersurgery compared to baseline in all groups (except sham-operated mice).Mice receiving the XMA052 MG1K antibody had smaller increase in LVEDD,LVESD and smaller decrease in LVFS compared to controls (FIG. 4).

The number of akinetic segments, a surrogate for infarct size, was3.9±0.4 in the saline-treated mice, and it was not affected by treatment(FIG. 5). Accordingly, the anterior wall (infarct) thickness was0.52±0.05 mm in the saline-treated and unaffected by treatment (FIG. 5).The MPI or Tei index, a marker of combined systolic and diastolicdysfunction and a surrogate marker for heart failure related mortality,was significantly increased after AMI (reflecting poor function) andpreserved in the mice treated with the XMA052 MG1K antibody (FIG. 5).Similarly, the TAPSE, a marker of right ventricular function and asurrogate marker for AMI related mortality, was significantly decreasedafter AMI (reflecting poor function) and partially preserved in the micetreated with the XMA052 MG1K antibody (FIG. 5). Thus, blockade of IL-1βusing the antibody ameliorates cardiac enlargement and dysfunctionfollowing AMI in the mouse, independent of infarct size. Pretreatmentwith an additional dose of the XMA052 MG1K antibody 48 hours prior tosurgery offered no advantage over treatment after surgery in this animalmodel (data not shown).

Example 5 Evaluation of an IL-1β Antibody in a Cardiovascular EventModel (Stroke)

Rodent (e.g., mice, rats) models of stoke may be used to evaluate theeffect of an IL-1β antibody or binding fragment thereof. For example, inone model, adult male Fischer rats are used (see for example, Morales etal., 2008, Circulation 118:1450-1459). In another model, C57BL/6 miceare used (see for example, Royl et al., 2009, Brain Res. 1265:148-157).Experiments are performed in a randomized fashion by investigatorsblinded to treatment groups. Permanent focal cerebral ischemia isinduced by occlusion of the middle cerebral artery (MCAO), such as bycauterization or monofilament occlusion. Rats/mice in which the MCA wasexposed but not occluded serve as sham-operated controls.

Control animal groups and MCAO groups then receive the treatmentantibody or placebo (e.g., control antibody) administeredintraperitoneally or intravenously at one or more pre-determined timesfollowing the procedure. For example, in one group the antibody isadministered immediately following the procedure and in another group,the antibody is administered 24 hours later.

In-life MCAO Antibody Txt Tests MRI Histology Group 1 Sham N/A Yes YesYes Group 2 Yes Placebo Yes Yes Yes Group 3 Yes Low dose (t = 0) Yes YesYes Group 4 Yes High dose (t = 0) Yes Yes Yes Group 3 Yes Low dose (24hr) Yes Yes Yes Group 4 Yes High dose (24 hr) Yes Yes Yes

Animals are evaluated for survival and body weight changes, as well asfunctional recovery (e.g., sensorimotor, behavioral testing, such aspole test, wire hanging test and/or neurological deficit score) andmeasurement of brain lesion size using MRI during the in-life stage(e.g., T2-weighted MRI), followed by histological examination (e.g., HEstaining and GFAP staining of coronal brain cryostat sections)post-sacrifice (e.g., at 4 weeks). Additionally, a computer-assistedhemisphere volumetry may be performed, based on T2-weighted MRI andHE-stained coronal brain cryostat sections. Additional test groups maybe evaluated to determine the effect on acute reperfusion after MCAO bymeasuring hemispheric cerebral blood flow with MRI (e.g., FAIR MRI).

Example 6 Evaluation of an IL-1β Antibody in a Model of PeripheralVascular Disease

To determine the effect of an IL-1β antibody or binding fragment thereofon peripheral vascular disease, an animal model of limb ischemia may beused (see for example, Park et al., Endocrinology 149:483-491, 2008).For example, limb ischemia is induced in C57BL/6 male mice by theligation of one femoral artery in anesthetized animals. Mice in whichthe artery is exposed but not ligated serve as sham-operated controls.

Control animal groups and artery ligation groups then receive thetreatment antibody or placebo (e.g., control antibody) administeredintraperitoneally or intravenously at one or more pre-determined timesfollowing the procedure. For example, in one group the antibody isadministered immediately following the procedure and in another group,the antibody is administered 24 hours later.

Ligation Antibody Txt LDPI Histology Group 1 Sham N/A Yes Yes Group 2Yes Placebo Yes Yes Group 3 Yes Low dose (t = 0) Yes Yes Group 4 YesHigh dose (t = 0) Yes Yes Group 3 Yes Low dose (24 hr) Yes Yes Group 4Yes High dose (24 hr) Yes Yes

The blood flow in both hind legs is assessed with a laser Dopplerperfusion image (LDPI) analyzer (Moor Instruments, Devon, UK), and theblood flow recovery is assessed by the ischemic limb to normal limbratio of blood flow. Serial blood flow measurements by LDPI are observedat regular intervals (e.g., daily for two weeks). Mice are euthanizedand the ischemic hind limb isolated for histological analysis.

After fixation with 4% paraformaldehyde, ischemic lower legs areembedded in OCT compound and frozen for cryostat sectioning. Tissuesections are stained with rat anti-mouse platelet EC adhesion molecule-1(PECAM-1) (PharMingen), mouse anti-α smooth muscle actin (SMA) (Sigma),and rat anti-mouse CD45 (PharMingen), rabbit anti-cGKI (Calbiochem). Toassess capillary density and inflammation, four random fields on twodifferent sections (≈3 mm apart) from each mouse are photographed and bycomputer-assisted analysis, capillary density is calculated as the meannumber of capillaries stained with PECAM-1 (endothelial marker) or a SMA(vascular smooth muscle marker). The mean number of infiltratingCD45-positive leukocytes is counted as the assessment of inflammation.

Example 7 Evaluation of an IL-1β Antibody in a Model of Atherosclerosis

The effect of an IL-1β antibody (XOMA 052) on macrophage-inducedcytokine production from endothelial cells and smooth muscle cells wasevaluated in a co-culture system. In this model, THP-1 cells werepre-activated to a macrophage-like phenotype with 200 nM PMA for 12hours, washed once and added to pre-plated human umbilical veinendothelial cells (HUVEC) or human coronary artery smooth muscle cells(CASMC) at a ratio of (10:1; 10⁶ THP-1 and 10⁵ HUVEC or CASMC) in thepresence or absence of XOMA 052, as indicated. Alternatively, cells wereincubated with rhIL-1β (R&D Systems) in the presence or absence of XOMA052, as indicated. After 48 hours, supernatants were removed andassessed for cytokine or enzyme content by ELISA (R&D Systems). Allassays were performed in triplicate. The data demonstrate that XOMA 052inhibits the release of IL-1β-induced pro-inflammatory molecules, suchas IL-6, IL-8, MCP-1 and PAI-1 from endothelial cells (p<0.05, FIG. 6,left panel). In addition, the data show that XOMA 052 inhibits therelease of IL-6 and IL-8 from smooth muscle cells, as well asIL-1β-driven MMP-3 and MMP-9 (p<0.05, FIG. 7, left panel). Importantly,it was also observed that XOMA 052 potently reduces the induction ofthese factors in the context of macrophage/EC or macrophage/SMCco-culture systems (p<0.05, FIGS. 6 & 7, right panel).

The ApoE knockout mouse is a well validated model of atherosclerosisthat follows a similar pattern of progression to that of human. MaleApoE^(−/−) mice on a C57BL/6 background were fed an atherogenic diet for16 weeks beginning at 6 weeks and treated with an IL-1β antibody, XMA052MG1K (i.p., twice weekly as indicated), control mouse IgG (i.p., twiceweekly, 1.0 mg/kg; Jackson ImmunoResearch), or quinapril (subQ, 10mg/kg, daily) for the duration of the study. En face analysis wascarried out using Sudan IV staining as described previously (Calkin etal., 2007, Atherosclerosis 195:17-22) and as follows. Aortas weredivided into arch, thoracic and abdominal aorta then cut longitudinally.After pinning en face onto wax, aortas were photographed and analyzed.Total and segmental plaque area was quantified as percentage areavisualized red as stained by Sudan IV. Aortas were subsequently embeddedin paraffin and sections cut for cross-sectional analysis.XMA052 MG1Kinhibited the formation of atherosclerotic lesions in ApoE knockout miceby 22-37% across the three doses tested (p<0.05, FIG. 8, 9).

Alternatively, plaque progression and in vivo coronary artery functionis assessed using noninvasive high-resolution ultrasound techniques (seefor example, Gronros et al., Am J Physiol Heart Circ Physiol.295:H2046-53, 2008). Eight-week-old male ApoE mice are fed a high-fatdiet with or without antibody treatment for approximately 16 weeks.During the course of treatment, total cholesterol levels are measured,as well as the degree of retardation of lesion progression in thebrachiocephalic artery, as visualized in vivo using an ultrasoundbiomicroscope. Histological analysis is also used to determine thereduction of brachiocephalic atherosclerosis. Coronary artery functionalso may be measured by volumetric flow, such as for example bysimultaneous recording of Doppler velocity signals and left coronaryartery morphology before and during adenosine infusion.

Antibody Txt Cholesterol Ultrasound Histology Group 1 Placebo Yes YesYes Group 2 Low dose Yes Yes Yes Group 3 Med dose Yes Yes Yes Group 4High dose Yes Yes Yes

To further characterize the impact of IL-1β antibody on the formation ofatherosclerotic lesions in the ApoE-knockout model, the aortic sinusand/or brachiocephalic artery is sectioned and assessed for lesioncross-sectional area and content (Zhou et al., 2008, Eur. J. Pharmacol.590:297-302; Calkin et al., 2007, Atherosclerosis 195:17-22; Kirii etal., 2003, Arterioscler. Thromb. Vasc. Biol. 23:656-660). Serial 3 μmparaffin sections are dewaxed and rehydrated. Endogenous peroxidaseactivity is inhibited by incubation with 3% hydrogen peroxide. Afterblocking sections with 20% (v/v) goat serum in phosphate-bufferedsaline, sections are incubated overnight at 4° C. with antibodiesagainst α-smooth muscle actin, inflammatory markers, such as IL-6, IL-8,MCP-1, ICAM-1 and VCAM-1, degradative enzymes, such as AMP-3, MMP-9 andcathepsin S or thrombotic factors, such as tissue factor or PAI-1.Sections are then incubated with the appropriate secondary antibodies.Positive areas are counted and expressed as a percentage of the wholeplaque area. A negative control, in which the primary antibody isreplaced with either mouse or rat IgG at the same dilution, is included.Sections are also evaluated for lipid content by staining with thelipophilic dye Oil Red 0 and macrophage infiltration is quantified byimmunohistochemistry by staining with antibodies against CD68 (Kirii etal., 2003, Arterioscler. Thromb. Vasc. Biol. 23:656-660). Blindedanalysis of positive immunostained sections is performed with animage-analysis program (Image Pro Plus, Media Cybernetics).

Alternatively, markers of inflammation and matrix degradation areinterrogated by quantitative gene expression analysis (Calkin et al.,2007, Atherosclerosis 195:17-22). RNA is extracted from whole aorta byhomogenization using Trizol and DNAse treated. Quantitative real timeRT-PCR is carried out using the Taqman system on an ABI Prism 7700Sequence Detector. Gene expression of the aforementioned genes arenormalized to 18S mRNA and reported as ratios compared to the level ofexpression in untreated control mice. For statistical purposes,non-parametric data are handled as their log derivative. Differences inexpression are compared using Student's t-tests (two groups) or one-wayANOVA (three or more groups).

The influence of IL-1β antibody on the aforementioned markers ofinflammation, degradation and thrombosis are also assessed in the serumof antibody-treated ApoE knockout mice by ELISA or using the MesoscaleDiscovery (MSD) platform. Serum obtained by cardiac puncture at the timeof sacrifice is analyzed for serum lipids as described (Warnick, 1986,Methods Enzymol. 129:101-23). All lipid assays are performed intriplicate determinations. An external control sample with known analyteconcentration is run for each assay to assure accuracy. Free plasmaglycerol concentrations is also determined and used to correct thetriglyceride values.

To quantitatively evaluate stability of atherosclerotic lesions,sections of 5 μm thickness are selected and quantified. Sections areserially cut every 50 μm from the cardiac base cross-section until theascending aorta appears. Approximately six serial 5-μm sections permouse are used for morphometric and immunohistochemical analysis.Collagen and foam cells in plaques are stained with a modified Movatpentachrome stain. Stained sections are inspected for buried fibrouscaps within the plaque, which are also counted. Morphometry is performedwith a computerized image-analysis program (Image Pro Plus, MediaCybernetics). Plaque composition, including extracellular lipids, foamcells and collagen is determined as a percentage of plaque area. Theplaque area is measured directly and subtracted from the area enclosedby the internal elastic lamina to derive the patent lumen area correctedby dividing internal elastic lamina surrounding area. The effect ofIL-1β antibody on plaque stability is evaluated by calculating thevulnerability index ((foam cells+extracellular lipids)/(collagens+smoothmuscle cells)) and the average number of buried fibrous caps.

Systolic and diastolic blood pressure are measured using a tail-cuffsystem and mean blood pressure calculated (Chamberlain et al., 2009,PLoS ONE 4(4): e5073). To ensure stress levels of mice are kept to aminimum, a single handler is used throughout the experiment and mice aresubjected to one week of training (blood pressure and pulse readings aretaken, but the data discarded) prior to starting analysis. Measurementsare taken at the same time, daily to avoid normal daily variance inblood pressure. In addition, the blood pressure is taken on the samepart of the tail every day. During analysis, 10 measurements are takeneach day, and mean blood pressure and standard deviation calculated foreach ‘data day’ and week (total of 50 readings per mouse per week, 10per day). On each day, individual data points are rejected if the bloodpressure is below 40 or above 210 mmHg, or if it is outside of 2standard deviations from the mean. All data for a day is rejected ifthere were less than 4 valid readings. Data for a week is rejected if itdoes not have at least 3 valid days of measurements. One week ofbaseline readings on chow diet are taken for each mouse, prior tofeeding of Western or WHC diets. Data are analyzed by global non-linearregression. This statistical test analyzes an entire family of data setssimultaneously sharing one or more parameters between data sets. Foreach shared parameter, global non-linear regression finds one best-fitvalue that applies to all the data sets. In this case, blood pressure isdetermined under control (chow fed) and treated (diet-fed) conditions,for different mouse genotypes, and global non-linear regressiondetermines whether the difference between each blood pressure curve isconvincing. The test does not compare individual time points, butinstead treats the data globally to produce a single p value percomparison.

These studies are further extended to evaluate the effect of the IL-1βantibody or fragment thereof on plaque rupture in carotid artery lesionsin the ApoE deficient murine atherosclerosis model (see for example,Nakamura et al., Atherosclerosis, 2009, Feb. 21 [Epub ahead of print]).ApoE-deficient 8-week-old mice (C57BL/6) are anesthetized and subjectedto ligation of the left common carotid artery just proximal to itsbifurcation. Four weeks after ligation, a polyethylene cuff is appliedjust proximal to the ligated site. Control groups are included in whichthe artery is exposed but not ligated, as well as ligated but notsubjected to the polyethylene cuff.

Animals then receive the treatment antibody or placebo (e.g., controlantibody) administered intraperitoneally or intravenously at one or morepre-determined times following the procedure. For example, in one groupthe antibody is administered 24 or 48 hours preceding cuff placement. Inanother group, the antibody is administered at the time of cuffplacement.

Ligation Cuff Antibody Txt Histology Group 1 Sham N/A N/A Yes Group 2Yes No N/A Yes Group 3 Yes No Placebo day 0 Group 4 Yes Yes Placebo day4 Group 5 Yes No Low dose (−24 hr) day 0 Group 6 Yes No High dose (−24hr) day 0 Group 7 Yes Yes Low dose (−24 hr) day 4 Group 8 Yes Yes Highdose (−24 hr) day 4 Group 9 Yes Yes Low dose (Day 0) day 4 Group 10 YesYes High dose (Day 0) day 4

Just before cuff placement (Day 0) and 4 days after cuff placement, miceare perfused through the left cardiac ventricle with isotonic saline and4% paraformaldehyde in 0.01 M phosphate buffer (pH 7.4) underphysiological pressure. Carotid arteries are collected and processed forhistological analysis. Cross-cryosections (6 μm) are prepared from theintracuff region of each carotid artery and stained with hematoxylin andeosin (H&E), and picrosirius red for collagen. The correspondingsections on separate slides are used for immunohistochemical stainingwith antibodies against neutrophils.

The proportions of intraplaque hemorrhage and disruption in theneointima accompanying the intramural thrombus are compared between theantibody and control groups. Histological classification of the plaquedisruption at the intracuff region of the carotid artery is done bydividing the lesions into three groups, based on the analyses of 30sections at 60 μm intervals in each sample tissue. When there are nocracks and no mural or occlusive thrombus at the intracuff region,classification is “no disruption”. When intraplaque hemorrhage, or muralor occlusive thrombus with cracks or erosion in the plaques aredetected, classified is “hemorrhage” or “disruption”, respectively.

Neutrophil infiltration in the neointima and collagen content is alsodetermined. Collagen content is evaluated by the picrosirius red-stainedpositive area which appears bright when viewed with polarized light.Neutrophil infiltration in the intima is assessed by the neutrophilspositive area which was stained by anti-neutrophil antibody (1:50;Serotec, MCA771GA).

Example 8 Cardiovascular Event Reduction in Subjects with a History ofat Least One Risk Factor for Cardiovascular Disease

To determine the effect of an IL-1β antibody or binding fragment thereofon reducing a cardiovascular event (e.g., time to first event) insubjects with a history of at least one risk factor for cardiovasculardisease, a clinical study is performed. In one study, an IL-1β antibodyis evaluated in an at risk population, measuring reduction of (e.g.,preventing) a primary outcome that includes a composite of death fromcardiovascular causes, myocardial infarction, or stroke, as well as eachoutcome separately. Measurements of reduction of (e.g., preventing) asecondary outcome may include death from any cause, the need for arevascularization procedure, heart failure, angina (e.g.,hospitalization for angina, unstable angina), congestive heart failure,and acute coronary syndrome.

For a double-blind study, subjects are randomly enrolled into one of twoIL-1β antibody treatment dose groups (e.g., 0.3 mg/kg, 0.1 mg/kg), or amatching placebo group. Antibody and placebo treatments are administeredin conjunction with standard of care. Men and women of at least 55 yearsin age are included in the study if they have a history of coronaryartery disease (e.g., manifest coronary artery disease), peripheralvascular disease, Type 2 diabetes, elevated total cholesterol,hypertension, low HDL cholesterol levels, tobacco smoking,atherosclerosis and/or microalbuminuria. Subjects are excluded if theyare known to have experienced a recent (e.g., within 6 months ofenrollment) cardiovascular event. Group sizes include sufficient numbersof subjects to detect a reduction in the relative risk of acardiovascular event during the period of the study. All subjectsprovide written informed consent.

Subjects are administered the IL-1β antibody or placebo at monthlyintervals and outcomes monitored throughout the study period (e.g., 3year study period). Outcomes are determined by standard clinicaldiagnoses accepted by the medical field. Results indicative of an effectfrom the IL-1β antibody include a reduction in the relative risk of acardiovascular event outcome (e.g., 20 percent reduction in relativerisk).

Example 9 Cardiovascular Event Reduction in Subjects with a History aPrevious Cardiovascular Event

To determine the effect of an IL-1β antibody or binding fragment thereofon reducing a cardiovascular event (e.g., time to second event) insubjects with a history of a previous cardiovascular event, a clinicalstudy is performed. In one study, an IL-1β antibody is evaluated insubjects in the period after the occurrence of a first documentedcardiovascular event of myocardial infarction or acute coronarysyndrome. The study measures reduction of (e.g., preventing) a primarycardiovascular event outcome that includes a composite of death fromcardiovascular causes, myocardial infarction, or stroke, as well as eachoutcome separately. Measurements for reduction of (e.g., preventing) asecondary outcome may include death from any cause, the need for arevascularization procedure, heart failure, angina (e.g.,hospitalization for angina, unstable angina), congestive heart failure,and acute coronary syndrome.

For a double-blind study, subjects are randomly enrolled into one of twodose groups (e.g., 0.3 mg/kg, 0.1 mg/kg) for an IL-1β antibody, or amatching placebo group. Men and women are enrolled in the studyfollowing a recent occurrence of a first cardiovascular event (e.g.,within 96 hours), as described above. Group sizes include sufficientnumbers of subjects to detect a reduction in the relative risk of asubsequent cardiovascular event during the period of the study. Allsubjects provide written informed consent.

Subjects are administered the IL-1β antibody or placebo at monthlyintervals and outcomes monitored throughout the study period (e.g., 3year study period). Outcomes are determined by standard clinicaldiagnoses accepted by the medical field. Results indicative of an effectfrom the IL-1β antibody include a reduction in the relative risk of asecond cardiovascular event outcome (e.g., 20 percent reduction inrelative risk).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Wherever an open-ended term isused to describe a feature or element of the invention, it isspecifically contemplated that a closed-ended term can be used in placeof the open-ended term without departing from the spirit and scope ofthe invention. Recitation of ranges of values herein are merely intendedto serve as a shorthand method of referring individually to eachseparate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. All methods described hereincan be performed in any suitable order unless otherwise indicated hereinor otherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseworking in the art upon reading the foregoing description. The inventorsexpect skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1. A method of reducing a cardiovascular event in a subject, comprisingadministering to said subject a therapeutically effective amount of ananti-IL-1β binding antibody or binding fragment thereof, wherein thesubject is a subject with a history of a previous cardiovascular eventor a history of at least one risk factor for cardiovascular disease, andwherein the cardiovascular event is myocardial infarction, stroke,cardiovascular death, congestive heart failure, cardiac arrest, acutecoronary syndrome, angina, or a revascularization procedure.
 2. Themethod of claim 1, wherein the subject is a subject with a history of aprevious cardiovascular event, and wherein the cardiovascular event ismyocardial infarction, stroke, acute coronary syndrome, angina or arevascularization procedure.
 3. The method of claim 1, wherein therevascularization procedure is a coronary, carotid or peripheralarterial revascularization procedure.
 4. The method of claim 3, whereinthe coronary, carotid or peripheral arterial revascularization procedureis a percutaneous coronary intervention (PCI), a stent implant, coronaryartery bypass graft (CABG), carotid endarterectomy, peripheral vasculardisease bypass surgery, or peripheral angioplasty surgery.
 5. The methodof claim 2, wherein said subject also has a history of at least one riskfactor for cardiovascular disease.
 6. The method of claim 1, wherein therisk factor is manifest coronary heart disease, coronary artery disease,thrombosis, transient ischaemic attack, left ventricular hypertrophy,arteriosclerosis, restenosis, tobacco smoking or peripheral vasculardisease.
 7. The method of claim 1, wherein the risk factor is elevatedtriglycerides, systemic inflammation, high blood phosphorus levels, highparathyroid hormone levels, microalbuminuria, or high homocysteinelevels.
 8. The method of claim 1, wherein the risk factor is obesity,hyperglycemia, chronic renal failure, high blood glucose, chronic kidneydisease, or metabolic syndrome.
 9. The method of claim 1, wherein therisk factor is hypertension, dyslipidemia, hyperlipidemia, elevatedtotal cholesterol, elevated LDL cholesterol, or low HDL cholesterol oratherosclerosis.
 10. The method of claim 1, wherein the subject haselevated levels of C-reactive protein (CRP).
 11. The method of claim 1,wherein the subject is older than 55 years.
 12. The method of claim 1,wherein the subject is non-hypertensive.
 13. The method of claim 1,wherein the subject has diabetes mellitus.
 14. The method of claim 13,wherein said diabetes mellitus is Type 2 diabetes.
 15. The method ofclaim 1, wherein the subject has a history of two or more said riskfactors.
 16. The method of claim 15, wherein the subject has a historyof three or more said risk factors.
 17. The method of claim 1, whereinadministering said therapeutically effective amount of an anti-IL-1βbinding antibody or binding fragment thereof is sufficient to achieve adecrease in CRP levels.
 18. A method of reducing mortality following acardiovascular event in a subject, comprising administering to saidsubject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof.
 19. The method of claim 18,wherein the cardiovascular event is myocardial infarction.
 20. Themethod of claim 18, wherein the cardiovascular event is stroke.
 21. Themethod of claim 18, wherein the cardiovascular event is congestive heartfailure.
 22. The method of claim 18, wherein the cardiovascular event isacute coronary syndrome.
 23. The method of claim 18, wherein thecardiovascular event is angina.
 24. The method of claim 18, wherein thecardiovascular event is a revascularization procedure.
 25. The method ofclaim 24, wherein the revascularization procedure is a coronary, carotidor peripheral arterial revascularization procedure.
 26. The method ofclaim 25, wherein the coronary, carotid or peripheral arterialrevascularization procedure is a percutaneous coronary intervention(PCI), a stent implant, coronary artery bypass graft (CABG), carotidendarterectomy, peripheral vascular disease bypass surgery, orperipheral angioplasty surgery.
 27. The method of claim 18, wherein thesubject does not have Type 2 diabetes.
 28. The method of claim 18,wherein the subject has survived a previous cardiovascular event ofmyocardial infarction or stroke.
 29. The method of claim 18, wherein theoccurrence of said cardiovascular event is a reoccurrence of acardiovascular event of myocardial infarction or stroke.
 30. The methodof claim 18, wherein the subject has a history of one or more riskfactors for cardiovascular disease.
 31. The method of claim 30, whereinthe risk factor is manifest coronary heart disease, coronary arterydisease, thrombosis, transient ischaemic attack, left ventricularhypertrophy, arteriosclerosis, restenosis, tobacco smoking or peripheralvascular disease.
 32. The method of claim 30, wherein the risk factor iselevated triglycerides, systemic inflammation, high blood phosphoruslevels, high parathyroid hormone levels, microalbuminuria, or highhomocysteine levels.
 33. The method of claim 30, wherein the risk factoris obesity, hyperglycemia, chronic renal failure, high blood glucose,chronic kidney disease, or metabolic syndrome.
 34. The method of claim30, wherein the risk factor is hypertension, dyslipidemia,hyperlipidemia, elevated total cholesterol, elevated LDL cholesterol, orlow HDL cholesterol or atherosclerosis.
 35. The method of claim 18,wherein the subject is non-hypertensive.
 36. The method of claim 30,wherein the subject has a history of two or more said risk factors. 37.The method of claim 36, wherein the subject has a history of three ormore said risk factors.
 38. The method of claim 18, whereinadministering said therapeutically effective amount of an anti-IL-1βbinding antibody or binding fragment thereof is sufficient to achieve adecrease in CRP levels.
 39. A method of reducing a cardiovascular eventin a subject with a history of at least one risk factor forcardiovascular disease, comprising administering to said subject atherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof, and wherein said risk factor is not Type 2diabetes, obesity, hyperglycemia, dyslipidemia, hyperlipidemia, chronicrenal failure, high blood glucose, chronic kidney disease, hypertension,atherosclerosis or metabolic syndrome.
 40. The method of claim 39,wherein the risk factor is not Type 2 diabetes.
 41. The method of claim39, wherein the cardiovascular event is myocardial infarction.
 42. Themethod of claim 39, wherein the cardiovascular event is stroke.
 43. Themethod of claim 39, wherein the cardiovascular event is cardiac arrest.44. The method of claim 39, wherein the cardiovascular event iscongestive heart failure.
 45. The method of claim 39, wherein thecardiovascular event is cardiovascular death.
 46. The method of claim39, wherein the cardiovascular event is acute coronary syndrome.
 47. Themethod of claim 39, wherein the cardiovascular event is angina.
 48. Themethod of claim 39, wherein the cardiovascular event is arevascularization procedure.
 49. The method of claim 48, wherein therevascularization procedure is a coronary, carotid or peripheralarterial revascularization procedure.
 50. The method of claim 49,wherein the coronary, carotid or peripheral arterial revascularizationprocedure is a percutaneous coronary intervention (PCI), a stentimplant, coronary artery bypass graft (CABG), carotid endarterectomy,peripheral vascular disease bypass surgery, or peripheral angioplastysurgery.
 51. The method of claim 39, wherein the risk factor is manifestcoronary heart disease, coronary artery disease, thrombosis, transientischaemic attack, left ventricular hypertrophy, arteriosclerosis,restenosis, tobacco smoking or peripheral vascular disease.
 52. Themethod of claim 39, wherein the risk factor is elevated triglycerides,systemic inflammation, high blood phosphorus levels, high parathyroidhormone levels, microalbuminuria, or high homocysteine levels.
 53. Themethod of claim 39, wherein the subject has elevated levels ofC-reactive protein (CRP).
 54. The method of claim 39, wherein thesubject is older than 55 years.
 55. The method of claim 39, wherein thesubject has a history of two or more said risk factors.
 56. The methodof claim 55, wherein the subject has a history of three or more saidrisk factors.
 57. The method of claim 39, wherein administering saidtherapeutically effective amount of an anti-IL-1β binding antibody orbinding fragment thereof is sufficient to achieve a decrease in CRPlevels.
 58. A method of treating a cardiovascular event in a subject,wherein the cardiovascular event is myocardial infarction, stroke,congestive heart failure, acute coronary syndrome or angina, comprisingadministering to said subject a therapeutically effective amount of ananti-IL-1β binding antibody or binding fragment thereof and at least oneother pharmaceutical composition comprising an active agent other thanan IL-1β antibody or fragment.
 59. The method of claim 58, wherein theactive agent of said at least one other pharmaceutical composition is acholesterol lowering agent, a statin, an HMG-CoA reductase inhibitor, acalcium channel blocker, a beta blocker, an antihypertensive, adiuretic, aspirin, niacin, an angiotensin-converting enzyme (ACE)inhibitor, an angiotensin II receptor blocker, a vasodilator, ananticoagulant, a inhibitor of platelet aggregation, a thrombolytic ordigitalis.
 60. A method for treating a cardiovascular event in asubject, wherein the cardiovascular event is myocardial infarction,stroke, congestive heart failure, acute coronary syndrome or angina,comprising administering to said subject a therapeutically effectiveamount of an anti-IL-1β binding antibody or binding fragment thereof anda revascularization procedure.
 61. The method of claim 60, wherein therevascularization procedure is a coronary, carotid or peripheralarterial revascularization procedure.
 62. A method of reducingrestenosis in a subject following a revascularization procedure,comprising administering to said subject a therapeutically effectiveamount of an anti-IL-1β binding antibody or binding fragment thereof.63. The method of claim 62, wherein the revascularization procedure is acoronary, carotid or peripheral arterial revascularization procedure.64. The method of claim 58, wherein administering said therapeuticallyeffective amount of an anti-IL-1β binding antibody or binding fragmentthereof is sufficient to achieve a decrease in CRP levels.
 65. A methodof treating acute hypertension in a subject comprising administering tosaid subject a therapeutically effective amount of an anti-IL-1β bindingantibody or binding fragment thereof and one or more antihypertensiveagents.
 66. The method of claim 65, wherein the subject has a bloodpressure of greater than or equal to 180/110 mm Hg.
 67. The method ofclaim 65, wherein the antihypertensive agent is administeredintravenously.
 68. The method of claim 65, wherein the antihypertensiveagent is labetalol, metoprolol, hydralazine, nitroglycerin, nicardipine,sodium nitroprusside or clevidipine.
 69. The method of any one of claim1, 18, 39, 58, 60, 62 or 65 further comprising administering at leastone other pharmaceutical composition comprising an active agent otherthan an anti-IL-1β binding antibody or binding fragment thereof. 70-78.(canceled)
 79. The method of any one of claim 1, 18, 39, 58, 60, 62 or65, wherein the anti-IL-1β binding antibody or binding fragment thereofbinds to human IL-1β with a dissociation constant of about 500 pM orless. 80-88. (canceled)
 89. The method of any one of claim 1, 18, 39,58, 60, 62 or 65, wherein the anti-IL-1β binding antibody or bindingfragment thereof is administered in one or more doses of 1 mg/kg or lessof antibody or fragment. 90-93. (canceled)
 94. The method of any one ofclaim 1, 18, 39, 58, 60, 62 or 65, wherein the anti-IL-1β bindingantibody or binding fragment thereof is administered as a fixed dose,independent of a dose per subject weight ratio. 95-100. (canceled) 101.The method of any one of claim 1, 18, 39, 58, 60, 62 or 65, wherein theanti-IL-1β binding antibody or binding fragment thereof is administeredby subcutaneous, intravenous or intramuscular injection.
 102. The methodof any one of claim 1, 18, 39, 58, 60, 62 or 65, wherein administrationof an initial dose of anti-IL-1β binding antibody or binding fragmentthereof is followed by the administration of one or more subsequentdoses. 103-107. (canceled)